Dose-dependent Antiproliferative Effect Research Articles

Antiproliferative Effect of Ornithogalum balansae on Human Cancer Cell Lines

The purpose of this study was to investigate the antiproliferative effect of the methanol and ethyl acetate extracts of Ornithogalum balansae flower and bulb against normal ARPE-19 cell line and cancer cell lines, A549, CRL-2923, HT-29 and HeLa. The cytotoxic activities of the extracts were determined using cell viability, deoxyribonucleic acid fragmentation and caspase-3 activity tests. Flower and bulb extracts exhibited dose-dependent antiproliferative effects on non-transformed and transformed cells, with greater growth inhibition in A549 lung cancer cells at lower concentrations. The IC50 values of the flower extracts against A549 cells were 1.5±0.153 and 2.2±0.503 µg/ml, and of the bulb extracts were 1.8±0.220 and 1.56±0.106 µg/ml, respectively. Against ARPE-19 cell line the IC50 values were 22.5±6.466 and 25±2.887 µg/ml for flower and 14±4.726 and 12.5±3.182 µg/ml for bulb extracts, respectively. In addition, it was observed that at concentrations ≤3.12 µg/ml there were statistically significant differences between ARPE-19 and transformed A549 cell lines (p <0.05). Finding caspase-3 activity of the methanol flower extract in A549 indicated apoptotic effects in those cells. These results showed that Ornithogalum balansae extracts could exhibit antitumor activity particularly on the A549 lung cancer cell line, a resistant cell line. Further investigations of the composition of the extracts are required to identify the effective components with antiproliferative activity, particularly on A549, the human alveolar adenocarcinoma cancer cell line. This study revealed that extracts of Ornithogalum balansae possessed considerable antiproliferative activity against A549 cells.

Sertraline in combination with sorafenib: A promising pharmacotherapy to target both depressive disorders and hepatocellular cancer

Hepatocellular carcinoma (HCC) is found within the first five most common tumors worldwide. Sorafenib is an approved agent in HCC treatment. Sertraline is a selective serotonin reuptake inhibitor. The aim of the study is to investigate the combination of sertraline and sorafenib at hepatocellular cancer cell proliferation and death. HepG2 cells were treated with drugs and viability test XTT was performed. Cells were stained with hematoxylin and eosin for histological examination or with anti-Bcl-2 antibody and Hoechst 33258 for immunofluorescence. Viability results supported dose-dependent antiproliferative effect for both sertraline and sorafenib. Microscopic evaluation of stained cells exerts morphological changes. This is the first study to show that sorafenib and sertraline have synergistic effect in hepatocellular cancer.

The Vitamin E Derivative Gamma Tocotrienol Promotes Anti-Tumor Effects in Acute Myeloid Leukemia Cell Lines.

Acute myeloid leukemia (AML) is a blood cancer characterized by the formation of faulty defective myelogenous cells with morphological heterogeneity and cytogenic aberrations leading to a loss of their function. In an attempt to find an effective and safe AML treatment, vitamin E derivatives, including tocopherols were considered as potential anti-tumor compounds. Recently, other isoforms of vitamin E, namely tocotrienols have been proposed as potential potent anti-cancerous agents, displaying promising therapeutic effects in different cancer types. In this study we evaluated the anti-cancerous effects of γ-tocotrienol, on AML cell lines in vitro. For this purpose, AML cell lines incubated with γ-tocotrienol were examined for their viability, cell cycle status, apoptotic cell death, DNA fragmentation, production of reactive oxygen species and expression of proapoptotic proteins. Our results showed that γ-tocotrienol exhibits time and dose-dependent anti-proliferative, pro-apoptotic and antioxidant effects on U937 and KG-1 cell lines, through the upregulation of proteins involved in the intrinsic apoptotic pathway.

Hempseed protein hydrolysates' effects on the proliferation and induced oxidative stress in normal and cancer cell lines.

Food proteins from different sources can provide beneficial effects on human health by releasing the bioactive peptides that are integral part of their native structure. In this study, we tested the biological potential of hempseed protein hydrolysates (HPHs) obtained from hempseed cake protein isolate. The HPHs were prepared by enzyme hydrolysis using three different proteases of microbial origin: Alcalase®, Neutrase® and Protamex®. The antioxidant activity of the obtained hydrolysates was determined by oxygen radical absorbance capacity (ORAC) assay, while the proliferative effects on normal (HaCaT) and cancer (HeLa) cells were determined by the CellTiter 96® AQueous One Solution Reagent (MTS) assay. HPHs showed dose-dependent antiproliferative effects on HeLa cells and stimulatory effects on the proliferation of HaCaT cells. HPH obtained by Neutrase® (HPH-N) showed the highest antioxidant activity expressed as an ORAC value. The protective effect of HPH-N on H2O2-induced oxidative stress in normal and cancer cells was evaluated and 1mg/mL of HPH-N significantly reduced the formation of intracellular reactive oxygen species (ROS) in both cell lines. The obtained results indicate the benefits of HPHs as potential natural antioxidants for the food industry and contribute to the growing trend of utilizing hempseed by-products.

Multi-targeted potential of Pittosporum senacia Putt.: HPLC-ESI-MSn analysis, in silico docking, DNA protection, antimicrobial, enzyme inhibition, anti-cancer and apoptotic activity

Pittosporum senacia (PS) Putt. (Pittosporaceae), indigenous to the Mascarene Islands, is a common ingredient in traditional medicines. However, there is currently a dearth of studies to validate some of these traditional claims. Given the broad traditional uses of PS against several diseases, we aimed to provide a comprehensive insight into the biological and chemical profile of P. senacia. The antioxidant, enzyme inhibitory activity, anticancer, and phytochemical composition of the methanolic extract of P. senacia leaf extracts were studied. The possible interaction and binding mode of the most abundant phytochemicals were studied via in silico docking experiments on tyrosinase and α-glucosidase. The mechanism behind the cytotoxic property of P. senacia extract for MDA-MB-231 was also examined using different methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability test checking apoptosis-associated genes, and wound healing assays. Twenty-six compounds were identified, of which caffeoylquinic acid derivatives, ferulic acid derivative, cinnamoylquinic acid derivative and two other polyphenols (oleuropeine and isoramnetin glucoside) being abundant, have been tested using in silico studies, against α-glucosidase and tyrosinase. The extract (IC50 = 118.8 μg/ml) exhibited time and dose dependent anti-proliferative effect on human breast cancer cell line, MDA-MB-231. According to the expression profile of apoptosis inhibitors and apoptosis promoters genes, expression of Bax and Bak genes were significantly increased compared to Bcl-2 and Birc5 genes. Based on wound healing analysis, cell migration was inhibited after the application of the plant extract. The present findings suggested that PS might be a good candidate as sources of bioactive compounds for designing functional applications.

Abstract 3859: Targeting CDK9 and MCL-1 by a New CDK9/p-TEFB Inhibitor with and without 5-fluorouracil in esophageal adenocarcinoma

Abstract Background: CDK9 inhibitors are anti-tumorigenic against several solid tumors including esophageal adenocarcinoma (EAC). However, efficacy of CDK9 inhibitor as adjuvant to chemotherapy and target proteins shared between these two drugs are not known. Methods: Efficacy of a new CDK9 inhibitor, BAY1143572 with and without 5-fluorouracil (5-FU) on tumor growth was tested in vitro and xenograft models of EAC. We tested effects of these agents on MCL-1 in vitro. We also analyzed a possible role of MCL-1 as a predictor of survival in patients with EAC. Results: BAY1143572 had a dose-dependent anti-proliferative and pro-apoptotic effects in vitro. BAY1143572 and 5-FU had strong synergy in vitro. Median tumor volume of FLO1 or ESO26 xenografts in cohort treated with combination of two drugs was 82% or 55% smaller than the cohort treated with 5-FU alone, and 46 % or 33% smaller than the cohort treated with BAY114352 alone (p&amp;lt;0.05). BAY1143572 transcriptionally downregulated MCL-1 by inhibiting HIF-1α binding to the MCL-1 promoter. 5-FU enhanced BAY1143572 induced MCL-1 downregulation in vitro. Stable overexpression of MCL-1 reduced apoptosis by BAY1143572 and 5-FU. Higher tumor cell MCL-1 expression correlated with shorter overall survival in patients with EAC treated with chemoradiation and surgery. Conclusion: CDK9 inhibitor and 5-FU are synergistic and MCL-1 is a target of CDK9 inhibitor in EAC. Citation Format: zhimin Tong, Alicia Mejia, Omkara Veeranki, Anuj Verma, Arlene Correa, Viren Patel, Rashmi Dokey, Luisa Solis, Barbara Mino, Riham Kathkuda, Jaime Canales, Steven Lin, Sunil Krishnan, Scott Kopetz, Mariela Blum, Jaffer Ajani, Wayne Hofstetter, Dipen M Maru. Targeting CDK9 and MCL-1 by a New CDK9/p-TEFB Inhibitor with and without 5-fluorouracil in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3859.

Analyzing bioactive effects of the minor hop compound xanthohumol C on human breast cancer cells using quantitative proteomics.

Minor prenylated hop compounds have been attracting increasing attention due to their promising anticarcinogenic properties. Even after intensive purification from natural raw extracts, allocating certain activities to single compounds or complex interactions of the main compound with remaining impurities in very low concentration is difficult. In this study, dose-dependent antiproliferative and cytotoxic effects of the promising xanthohumol (XN) analogue xanthohumol C (XNC) were evaluated and compared to XN and a XN-enriched hop extract (XF). It was demonstrated that the cell growth inhibition of human breast cancer cell line (MCF-7) significantly increases after being treated with XNC compared to XN and XF. Based on label-free data-dependent acquisition proteomics, physiological influences on the proteome of MCF-7 cells were analyzed. Different modes of action between XNC and XN treated MCF-7 cells could be postulated. XNC causes ER stress and seems to be involved in cell-cell adhesion, whereas XN influences cell cycles and DNA replication as well as type I interferon signaling pathway. The results demonstrate the utility of using quantitative proteomics for bioactivity screenings of minor hop compounds and underscore the importance of isolating highly pure compounds into their distinct forms to analyze their different and possibly synergistic activities and modes of action.

Isolation of thymoquinone from &lt;i&gt;Nigella sativa&lt;/i&gt; L. and &lt;i&gt;Thymus vulgaris&lt;/i&gt; L., and its anti-proliferative effect on HeLa cancer cell lines

Purpose: To isolate thymoquinone (TQ) from Nigella sativa L. and Thymus vulgaris L., and investigate its anti-proliferative effect on HeLa cancer cells.Method: Pulverized dried samples of N. sativa seed (100 g) and aerial parts of T. vulgaris (1000 g) were subjected to Soxhlet extraction using methanol and n-hexane combined in different proportions. Thymoquinone (TQ) was then isolated from the extracts using high performance liquid chromatography (HPLC). The isolated TQ was further subjected to Fourier Transform Infrared (FTIR) spectroscopy to identify its functional groups. The anti-proliferative effect of TQ on HeLa cancer cells was evaluated using 3-[4, 5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay.Results: Extract yield from N. sativa was significantly higher than from T. vulgaris, and also increased with increase in the proportion of methanol in the extraction solvent (p &lt; 0.05). Methanol and n-hexane (4:1, v:v) yielded the highest amount of oil, with yields of 15.8 and 9.7 ml/25 g dry weight (d.wt.) from N. sativa and T. vulgaris, respectively. The results obtained from HPLC showed that the concentration of TQ isolated from N. sativa (388.61 μg/ml) was significantly higher than that from T. vulgaris (357.03 μg/ml, p &lt; 0.05). The anti-proliferative effects of TQ standard and TQ isolated from N. sativa on HeLa cancer cells were dose-dependent, and was highest at the lowest concentration. The number of viable cells significantly decreased with increase in TQ concentration (p &lt; 0.01). TQ from N. sativa significantly reduced the number of viable cells even at the lowest concentration when compared to TQ standard (p &lt; 0.05). Cell death was significantly higher in TQ-treated groups than in untreated cancer cells.Conclusion: The results obtained in this study show that N. sativa is a potential source of TQ, with the yield enhanced by modifying the extraction procedure or solvent used. Furthermore, TQ isolated from N. sativa exerts a dose-dependent anti-proliferative effect on HeLa cancer cells.Keywords: Thymoquinone, Nigella sativa, Thymus vulgaris, Anti-proliferative effect

Targeting CDK9 and MCL-1 by a new CDK9/p-TEFb inhibitor with and without 5-fluorouracil in esophageal adenocarcinoma.

Background:CDK9 inhibitors are antitumorigenic against solid tumors, including esophageal adenocarcinoma (EAC). However, efficacy of a CDK9 inhibitor combined with 5-fluorouracil (5-FU) and target proteins that are targeted by these agents in EAC are unknown.Methods:The anti-EAC efficacy of a new CDK9 inhibitor, BAY1143572, with and without 5-FU was assessed in vitro and in xenograft models in athymic nu/nu mice. Synergy between BAY1143572 and 5-FU in inhibiting cell proliferation was analyzed by calculating the combination index using CompuSyn software. Potential targets of BAY1143572 and 5-FU were identified by reverse-phase protein array. The effects of BAY1143572 and 5-FU on MCL-1 in vitro were analyzed by Western blotting, quantitative real-time polymerase chain reaction, and chromatin immunoprecipitation assay. MCL-1 protein expression in tumors from patients with locoregional EAC treated with chemoradiation and surgery was assessed by immunohistochemistry.Results:BAY1143572 had dose-dependent antiproliferative and proapoptotic effects and demonstrated synergy with 5-FU against EAC in vitro. The median volumes of FLO-1 and ESO-26 xenografts treated with 5-FU plus BAY114352 were significantly smaller than those of xenografts treated with either agent alone (p < 0.05). BAY1143572 downregulated MCL-1 by inhibiting HIF-1α binding to the MCL-1 promoter. 5-FU enhanced BAY1143572-induced MCL-1 downregulation and stable MCL-1 overexpression reduced the apoptosis induced by BAY1143572 and 5-FU in vitro. High patients’ tumor MCL-1 expression was correlated with shorter overall and recurrence-free survival.Conclusions:BAY1143572 and 5-FU have synergistic antitumorigenic effects against EAC. MCL-1 is a downstream target of CDK9 inhibitors and a predictor of response to neoadjuvant chemoradiation in EAC.

Methotrexate-induced apoptosis in human ovarian adenocarcinoma SKOV-3 cells via ROS-mediated bax/bcl-2-cyt-c release cascading.

IntroductionThe communication between a substance and a cell may depend on whether the cell is normal or pathological. The disease cells and drug interaction may occasionally overcome beneficial action of the drug; subsequently, it is important to investigate the effect of the drug in both the normal and target cells. This study aimed to evaluate the methotrexate (MTX) antiproliferative effect and explore the mechanistic approach to investigate the cell death index in SKOV-3 ovarian cells during treatment with MTX.MethodsIn vitro studies of SKOV-3 cells were examined by tetrazolium assay after exposure to various concentrations of MTX. Moreover, reactive oxygen species (ROS) generation, mitochondrial membrane potential, DNA damage, and AO/EtBr staining morphological analysis of necrotic/apoptotic cells were detected; cellular impairment in mitochondria and DNA was confirmed by JC-1 mitotracker/DAPI, respectively, and cell death pathway markers; bax/bcl-2 were analyzed.ResultsA dose-dependent antiproliferative effect of MTX treatment was observed in SKOV-3 cells; the prominent inhibitory concentration was 40 µM of MTX (P<0.01). The growth inhibition rates of the cancer cells reached 24.07% in MTX. The MTX showed increase in ROS generation and mitochondrial depolarization, and DNA integrity cells collectively advocated the apoptotic cell death at higher concentration. In addition, the results of reverse transcription polymerase chain reaction also supported the apoptosis by upregulating the bax and downregulating the bcl-2 (P<0.01). Thus the MTX moderately provokes apoptosis.ConclusionOur findings suggest that MTX acts on SKOV-3 cancer cells by increasing intracellular ROS levels, leading to DNA damage and altering the MMP along with apoptotic gene upregulation. This mechanism may provide new therapeutic targets to improve tumor treatment.

Functional Role of Linc-RNAs in Multiple Myeloma: Linc-MIR17HG Affects Fatty Acid Biosynthesis Via transcriptional Regulation of ACC1 with Potential Therapeutic Implications

Besides the well described function of RNA to produce proteins, a large volume of transcribed product has non-coding function. A recent analysis of RNA repertoire has identified a family of non-coding transcripts with sequence longer than 200 nucleotides, the long intergenic non-coding RNAs (lincRNAs). Although lincRNAs have been considered to provide regulatory functions, their precise role in cellular biology remains unclear. Using our RNA-seq data from 360 newly-diagnosed patients and 18 normal plasma cells, we have recently described the landscape of lincRNAs in multiple myeloma (MM) and reported their role as independent risk predictors for survival outcome.We have now studied the functional role of a lincRNA, the miR-17-92 primary precursor linc-MIR17HG, present in our lincRNA profile and highly correlated with overall survival in MM. We observe that inhibition of linc-MIR17HG by antisense LNA GapmeRs (n=2) leads to apoptosis in 12 genotypically distinct MM cell lines as well as in 13 primary patient MM cells. These effects are not fully rescued by expression of miR-17-92 microRNAs, suggesting a distinct biological function for linc-MIR17HG in MM. We therefore performed gene expression profile in 2 MM cell lines (AMO1 and NCI-H929) and in 2 primary patient MM cells after short-term suppression of linc-MIR17HG; and, at these early time points (18-36h), we found significant downregulation of a subset of genes (FC>2; p<0.05) including ACC1, EXT1, EPT1, ANO6, CCDC91 and KIA1109, but not miR-17-92 microRNAs. These transcriptional changes were validated by qRT-PCR in MM cell lines and primary MM cells exposed to different LNA GapmeRs targeting linc-MIR17HG with a non-overlapping spectrum of off-target effects. Importantly, our RNA-seq analysis of 360 newly-diagnosed MM patients from IFM/DFCI 2009 clinical study showed that expression of linc-MIR17HG strongly correlated with the expression of each of these genes (R2>0.4; p<0.01) in MM patients, further suggesting a regulatory function by linc-MIR17HG at transcriptional level. Using CRISPR interference (CRISPRi), we have also identified that the linc-MIR17HG with transcriptional regulatory functions is not produced from the canonical transcript isoforms MIR17HG-201/-203; these isoforms, rather, appear to be involved in production of microRNAs, leaving an alternative transcription start site usage as possible source for the transcriptional regulator isoform(s).We next investigated whether regulation of these early targets may contribute to the activity of linc-MIR17HG. We performed a RNAi-based screening in 2 MM cells lines (AMO1 and NCI-H929) by silencing each of the linc-MIR17HG downstream target genes with at least 2 different highly-specific siRNAs. This approach revealed that silencing of acetyl-CoA carboxylase 1 (ACC1, also known as ACACA), a gene encoding the limiting enzyme in the biosynthesis of fatty acids, significantly affects MM cell growth and viability. These results were validated using stable knock-down via shRNAs, confirming ACC1 as a novel vulnerability in MM. These results provide a molecular basis for reported role of fatty acid metabolism in MM cell growth and survival. We have now evaluated two orally available inhibitors of ACC1 activity, ND-630 and ND-646, in a panel of 10 MM cell lines, an report a potent time- and dose-dependent anti-proliferative effect. The activity of these inhibitors and linc-MIR17HG on fatty acid biosynthesis in MM cells is under investigation and will be presented.We have also begun to investigate molecular pathway used by linc-MIR17HG to modulate ACC1 function. Our preliminary data suggest that linc-MIR17HG may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity.In conclusion, we highlight a transcriptional regulatory activity of a lincRNA in MM with significant functional impact that can be therapeutically translated. DisclosuresAnderson:Bristol Myers Squibb: Consultancy; Millennium Takeda: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; OncoPep: Equity Ownership, Other: Scientific founder; C4 Therapeutics: Equity Ownership, Other: Scientific founder; Celgene: Consultancy. Munshi:OncoPep: Other: Board of director.

Mechanistic Insights of Pomalidomide Activity in Combination with Bortezomib and Dexamethasone in Multiple Myeloma and Immune Cells

Introduction: Results from the Phase III, randomized, open-label, clinical study OPTIMISMM comparing the combination of pomalidomide, bortezomib and low-dose dexamethasone (PVd) to bortezomib and low-dose dexamethasone (Vd) in patients with relapsed/refractory multiple myeloma who have all received 1-3 prior lines of therapy, demonstrated significant improvement in progression-free survival (PFS) for pomalidomide versus the comparator arm. Each agent exhibits cytotoxic effects on multiple myeloma (MM) cells when given alone, but acts synergistically in combination, due to non-redundant mechanisms of action. Furthermore, pomalidomide has immuno-stimulatory functionality and enhances T cell and NK-cell-mediated immunity. Pomalidomide binds Cereblon and facilitates degradation of Aiolos and Ikaros, conferring direct cytotoxic effects, while stimulating immune activity. MG132, a proteasome inhibitor (PI), can stabilize pomalidomide-mediated degradation of Aiolos and Ikaros at relatively high concentrations in vitro. A key question is whether PIs, at therapeutic levels, inhibit pomalidomide activity. One obstacle to achieving greater understanding of the bortezomib impact on pomalidomide activity is the highly cytotoxic effect of bortezomib on cultured MM cells. Following intravenous bortezomib injection at the maximal tolerated dose, the Cmax in blood is 260-520 nM, 5 minutes after injection, followed by a rapid drop-off. In vitro, bortezomib is cytotoxic at 1-4 nM, limiting the usefulness of the cell culture model. To circumvent this issue, we implemented a pulse method developed by Shabaneh (2013) and identified sublethal concentrations of bortezomib that had synergistic anti-proliferative effects at concentrations within the clinical window.Results: Pomalidomide alone had a dose-dependent anti-proliferative effect on all MM cell lines tested and addition of dexamethasone led to further inhibition of proliferation. Bortezomib pulse at concentrations ≥ 40 nM (MM1.S), 70 nM (H929) or 2 uM (LP-1) led to complete inhibition of proliferation at all concentrations of pomalidomide or dexamethasone used. The synergy and/or additivity of drug combinations was analyzed using the Chou-Talalay Method. Synergies of pomalidomide and bortezomib were observed in the 1-4 nM concentration range for continuous treatment, and 50-560 nM for pulsed treatment, with pomalidomide most synergistic around 100nM. Degradation of Ikaros, Aiolos and ZFP91 was confirmed in MM cells treated with pomalidomide alone and in combination with bortezomib, indicating no inhibitory effect of proteasomal inhibition on substrate degradation.In peripheral blood mononuclear cells (PBMCs), IL-2 and Granzyme B production was induced by pomalidomide and inhibited by bortezomib. The combination of pomalidomide and bortezomib (PV) also enhanced IL-2 and Granzyme B secretion albeit to a lesser extent than pomalidomide alone. Degradation of Aiolos, Ikaros and ZFP91 was also observed in isolated T-cells and results closely paralleled those of MM cells. T-cell proliferation was also enhanced by pomalidomide, with maximal effects observed in the 100 nM-1 uM concentration range. While bortezomib alone decreased T-cell proliferation by ~3-fold, the proliferation effect was comparable in cells treated with pomalidomide alone and PV.Conclusion: Pomalidomide, bortezomib and dexamethasone exert synergistic anti-proliferative effects on tumor cells. Despite inhibiting proteasomal activity, bortezomib does not antagonize pomalidomide-mediated degradation of Cereblon substrates Aiolos, Ikaros and ZFP91 when used at therapeutically relevant concentrations. Pulsing experiments provide the advantage of mimicking bortezomib dosing regimens; under these conditions, pomalidomide-mediated degradation of substrates was still observed. In immune cells, the PV combination has an overall positive impact on T-cell proliferation despite inhibitory activity of single agent bortezomib. Our results suggest that in combination, pomalidomide can overcome the immunosuppressive effects of bortezomib, when bortezomib is used at therapeutic levels. DisclosuresKatz:Celgene: Consultancy. Bjorklund:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership. Serbina:Celgene: Employment.

Isolation, Characterization and Antitumor Effect on DU145 Cells of a Main Polysaccharide in Pollen of Chinese Wolfberry.

Modern studies have shown that pollen has a certain role in the treatment of prostate-related diseases. In the present study, pollen polysaccharides from Chinese wolfberry (WPPs) were extracted by hot-water extraction and ethanol precipitation, further purified by chromatography on a DEAE-cellulose column and Sephadex G-100 column. Homogeneous polysaccharide CF1 of WPPS was obtained, the molecular weight of which was estimated to be 1540.10 ± 48.78 kDa by HPGPC-ELSD. HPLC with PMP derivatization analysis indicated that the monosaccharide compositions of CF1 were mannose, glucuronic acid, galacturonic acid, xylose, galactose, arabinose, and trehalose, in a molar ratio of 0.68:0.59:0.27:0.24:0.22:0.67:0.08. The antitumor effects of CF1 upon MTT, Tunel assay and flow cytometry assay were investigated in vitro. The results showed that CF1 exhibited a dose-dependent antiproliferative effect, with an IC50 value of 374.11 μg/mL against DU145 prostate cancer cells. Tunel assay and flow cytometry assay showed that the antitumor activity of CF1 was related to apoptosis in vitro. The present study suggested that the CF1 of WPPs might be a potential source of antitumor functional food or agent.

Magnetic nickel ferrite nanoparticles: Green synthesis by Urtica and therapeutic effect of frequency magnetic field on creating cytotoxic response in neural cell lines.

Despite of great attention concerned on Ni ferrite nanostructures in bioapplications, little is known about the toxicity of these NPs at the cellular and molecular levels. U87 (human primary glioblastoma) and SH-SY5Y (human neuroblastoma) cells treated with various concentration of well-characterized magnetic nickel ferrite nanoparticles, exposed to frequency magnetic field (FMF) and their response was studied. Ferromagnetic nanocrystalline nickel ferrite (NiFe2O4) powder that characterized by XRD, TEM, SEM, FT-IR, nanosizer, and VSM techniques were prepared by a hydrothermal method in the presence of Urtica plant extract as a green precursor that acts both as reducing and capping agent. Owing to the exceptional properties of green alkalinized agent such as minor toxicity, higher biodegradability, high active surface and environment compatibility, we used the green alkalinized agent (Utrica) to prepared nanostructures for the first time. According to the obtained results, the FMF exposure caused an increase in cell death in neural cell types 48 h after treatment. MNPs indicated dose-dependent cytotoxicity but the amount of cell death per cell in the absence of MFM for SH-SY5Y cells was more than in U87 cells. On the other hand, cell death induced by FMF exposure was observed specifically in SH-SY5Y cells. Nevertheless, it is essential to perform more investigations to find the exact related mechanisms. Imatinib showed dose-dependent antiproliferative effects in all three prostate cancer cell lines.

Synthesis, structural characterization, anti-proliferative and antimicrobial activity of binuclear and mononuclear Pt(II) complexes with perfluoroalkyl-heterocyclic ligands

In this paper we report the synthesis of four Pt(II) complexes with 5-perfluoroalkyl-1,2,4-oxadiazolyl-pyridine and 3-perfluoroalkyl-1-methyl-1,2,4-triazolyl-pyridine ligands. Two binuclear complexes [PtCl(pfibap)2](µ-Cl)2 (1), [Pt2(µ-Cl)2(pfioap)4]Cl2 (2), and two mononuclear [PtCl2(pfptp)] (3), [PtCl2(pfhtp)2] (4), were synthesized with the ligands: 2-(5-perfluoropropyl)-1,2,4-oxadiazole-3yl)-pyridine (pfpop), 2-(5-perfluoroheptyl-1,2,4-oxadiazole-3yl)-pyridine (pfhop), 2-(3-perfluoropropyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfptp), 2-(3-perfluoroheptyl-1-methyl-1,2,4-triazole-5yl)-pyridine (pfhtp), and were structurally characterized. All complexes were tested in vitro on three tumor cell lines MCF-7 (human breast cancer), Hela (epithelioid cervix carcinoma), HCT-116 (human colorectal carcinoma). Compounds 1 and 2 showed a dose-dependent anti-proliferative effect against the three tumor cell lines whereas they did not affect viability of intestinal normal-like differentiated Caco-2 cells. Results showed that both the compounds act as pro-apoptotic agents inducing a clear shift of viable cells towards early apoptosis, while they do not exert any necrotic effect. They also caused cell cycle perturbation with significant decrease in the percentage of cells in the G0/G1 phase, accompanied by a concomitant percentage increase of cells in the G2/M phase, and appearance of a subG1-cell population. Moreover, all complexes showed antimicrobial activity against Escherichia coli, Kocuria rhizophila and two Staphylococcus aureus strains and a different behavior in binding activity to DNA.

Lycosin-II Suppresses the Growth of Tumor Cells and Kills them Through Membrane Disruption and Apoptosis Induction

Recently, there has been a growing interest in the discovery of novel chemotherapeutic agents that have fewer side effects and are less likely to develop resistance. This study was conducted to examine the antitumor activity of lycosin-II, an antibacterial peptide isolated from the spider Lycosa singoriensis. Computational analysis revealed that lycosin-II has appropriate structural parameters (cationicity, α-helical conformation, amphipathicity) applicable for exhibiting antitumor activity. The results of MTT assay showed that lycosin-II possesses cytotoxicity (IC50 = 70.79 µg/ml) against the human colorectal cancer cell line HCT 116 as these cells were killed 30 min after treatment. Lycosin-II also exhibits a dose-dependent antiproliferative effect, as assessed using MTT assay coupled with direct cell counting as a measure of cell growth. Lactate dehydrogenase leakage assay and scanning electron microscopy revealed that lycosin-II kills tumor cells through cell membrane disruption. Apoptosis assays showed that Bax proapoptotic protein expression level and caspase-3 activity were increased after lycosin-II treatment, which indicates the activation of intrinsic apoptotic pathway. Altogether, these data indicate that lycosin-II suppresses the growth of tumor cells and kills them through membrane disruption and activation of apoptosis. However, lycosin-II exerts moderate hemolytic activity, which may be a major challenge in therapeutic applications. In conclusion, lycosin-II may serve as a template for designing novel antitumor peptides with low toxicity to normal cells for chemotherapeutic applications.

Three Dimensional Mixed-Cell Spheroids Mimic Stroma-Mediated Chemoresistance and Invasive Migration in hepatocellular carcinoma

Interactions between cancer cells and cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME) play an important role in promoting the profibrotic microenvironment and epithelial-mesenchymal transition (EMT), resulting in tumor progression and drug resistance in hepatocellular carcinoma (HCC). In the present study, we developed a mixed-cell spheroid model using Huh-7 HCC cells and LX-2 stellate cells to simulate the in vivo tumor environment with respect to tumor-CAF interactions. Spheroids were cultured from cancer cells alone (monospheroids) or as a mixture (mixed-cell spheroids) in ultra-low-attachment plates. Compact, well-mixed, and stroma-rich mixed-cell spheroids were successfully established with heterotypic cell-cell contacts shown by the presence of gap junctions and desmosomes. Mixed-cell spheroids showed enhanced expression of collagen type-I (Col‐I) and pro‐fibrotic factors such as, transforming growth factor beta1 (TGF-β1), and connective tissue growth factor (CTGF) compared to the levels expressed in mono-spheroids. The EMT phenotype was evident in mixed-cell spheroids as shown by the altered expression of E-cadherin and vimentin. Differential drug sensitivity was observed in mixed-cell spheroids, and only sorafenib and oxaliplatin showed dose-dependent antiproliferative effects. Simultaneous treatment with TGF-β inhibitors further improved sorafenib efficacy in the mixed-cell spheroids, indicating the involvement of TGF-β in the mechanism of sorafenib resistance. In 3D matrix invasion assay, mixed-cell spheroids exhibited fibroblast-led collective cell movement. Overall, our results provide evidence that mixed-cell spheroids formed with Huh-7 and LX-2 cells well represent HCC tumors and their TME in vivo and hence are useful in studying tumor-stroma interactions as mechanisms associated with drug resistance and increased cell motility.

Abstract 1967: Synthesis, in vitro evaluation in ovarian cancer cells and molecular modeling analysis of novel 2-aminothiazole and 2-aminopyridine derivatives

Abstract Background: Resistance to chemotherapy remains a major hurdle in complete cure against ovarian cancer. Among 75 percent of respondents to chemotherapy, the majority would relapse within two years. Cancer cells can overcome therapy-induced damage by activation of back-up signaling pathways and flexible modulation of affected networks resulting in chemo-resistance. Platinum-based drugs are the most active agents in use against the ovarian cancer. Non platinum-based chemotherapy regimens are usually given in the ‘platinum-resistant' setting. Methods: A series of N-protected and N-deprotected amino acid derivatives of ethyl-2-aminothiazole-4-carboxylate (4a-d &amp; 6a-d) and 2-aminopyridine (5a-d &amp; 7a-d) have been synthesized and characterized by FT-IR, 1H NMR, 13C NMR, mass spectrometry and elemental analysis. The compounds were evaluated in silico for selected target inhibition. AutoDockVina virtual screening program was used to target multiple markers in cell signaling pathway (including p110a/PI3K, EGFR, VEGFR, PDGFR, BCR-ABL, RET, c-KIT, c-Raf, ALK, ,B-Raf, , CTLA-4). Anti-tumor activity was determined in parent (A2780) and cisplatin resistant (A2780CISR) ovarian cancer cell lines by MTT reduction assay.Binding affinity data of standard drugs and synthesized compounds 4c, 6b and 7c were compared. Binding mode and 2D interactions of the compounds with p110a isoform of PI3K, EGFR, VEGFR and PDGFR were determined. Results: Among selected targets, p110a isoform of PI3K is the main target involved in development of ovarian cancer; all three ligands were found to be active against this target. Compound 4c&amp;6b exhibited excellent inhibition of p110a isoform of PI3K with binding energy -8.3 Kcal/mol and -8 Kcal/mol that is comparable to the reference compound i.e. -9.8 Kcal/mol. Overall, compound 4c&amp;6b exhibited greatest inhibition with lowest binding energies against most of the targets.All the tested compounds exhibited dose dependent anti-proliferative effect against both cell lines. IC50 and RF values of cisplatin and tested compounds were determined. Compounds 4c, 6b and 7c showed interesting results as they were more active in cisplatin resistant cell line than in parent cells. The RF values of these compounds were 0.83, 0.46 and 0.53 respectively. Discussion: RF value of a compound provides an idea if the compound can induce more cell death than corresponding drug in resistant cell lines. RF value less than 1 for a compound/drug is an indication of ability of that compound to bring about more cell death than standard drug in resistant cells. These data warrant further investigations into the mechanism of reversal of resistance induced by potential compounds. Citation Format: Shagufta Naz, Humaira Nadeem, sadia sarwar, Rehan Z. Paracha, Jun Q. Yu, Fazlul Huq. Synthesis, in vitro evaluation in ovarian cancer cells and molecular modeling analysis of novel 2-aminothiazole and 2-aminopyridine derivatives [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 1967.

Abstract 3188: Combination treatment strategies to enhance the effect of standard chemotherapeutic agents in Ewing sarcoma cells

Abstract EWS/FLI1 is a unique chimeric fusion protein found in approximately 85% of Ewing sarcoma (ES) tumors. Blocking this fusion protein in conjunction with administration of anticancer agents could be an effective strategy for treating ES. Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), also known as survivin, is overexpressed in several malignancies, including ES. Survivin is associated with aggressive growth of cancer and resistance to standard chemotherapy and thus serves as a prognostic marker for ES and other cancers. EWS/FLI1 and Specificity protein1 (Sp1) jointly mediate the expression of cyclins to facilitate cell growth. Mithramycin-A (Mit-A) has been identified as a potent inhibitor of EWS-FLI1 and its downstream targets. Preclinical research from our laboratory demonstrated that clotam, a small molecule, exhibits anticancer activity by targeting Sp1 and survivin. The objective of our investigation is to test the efficacy of Mit-A or clotam to induce the antiproliferative activity of standard chemotherapeutic agents, vincristine (Vin) or etoposide (Eto), in ES cell lines. TC71, TC32, CHLA32, CHLA10 and TC205 cells were treated with increasing concentrations of Mit-A/clotam/Vin/Eto. Cellular growth inhibition was evaluated at 48 h post-treatment using CellTiter-Glo kit (Promega). In order to determine the combination response, cell growth inhibition of each chemotherapeutic drug was assessed in the presence or absence of Mit-A or clotam. Further studies were performed to investigate the effect of individual agents and combinations on apoptosis and cell cycle phase distribution. Levels of cleaved poly (ADP-ribose) polymerase (c-PARP) were determined by Western blot analysis and the apoptotic cell population quantitated by flow cytometry analysis of Annexin-V stained cells. Cell cycle phase distribution was also measured by flow cytometry using propidium iodide. Results showed a dose-dependent antiproliferative effect with all agents. The combination of Mit-A or clotam with Vin or Eto showed greater effect on apoptosis and cell cycle arrest compared to the effects of Vin or Eto alone. Overall, these results indicate the potential clinical advantage of combination treatment involving Mit-A or clotam for inhibiting the growth of ES cells. Molecular profiling analysis is under way to elucidate the candidate pathways associated with beneficial effect of our combination treatments and with other chemotherapeutic agents. Citation Format: Anish Ray, Sagar Shelake, Umesh Sankpal, Lina Albeer, Holly Lout, Abigail Hunter, Kathryn Dunlap, Rajasekhar Maram, W. Paul Bowman, Riyaz M. Basha. Combination treatment strategies to enhance the effect of standard chemotherapeutic agents in Ewing sarcoma cells [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 3188.

Curcumin as Treatment for Bladder Cancer: A Preclinical Study of Cyclodextrin-Curcumin Complex and BCG as Intravesical Treatment in an Orthotopic Bladder Cancer Rat Model.

Objective To evaluate the antitumor effect of cyclodextrin-curcumin complex (CDC) on human and rat urothelial carcinoma cells in vitro and to evaluate the effect of intravesical instillations of CDC, BCG, and the combination in vivo in the AY-F344 orthotopic bladder cancer rat model. Curcumin has anticarcinogenic activity on urothelial carcinoma and is therefore under investigation for the treatment of non-muscle invasive bladder cancer. Curcumin and BCG share immunomodulating pathways against urothelial carcinoma. Methods Curcumin was complexed with cyclodextrin to improve solubility. Four human urothelial carcinoma cell lines and the AY-27 rat cell line were exposed to various concentrations of CDC in vitro. For the in vivo experiment, the AY-27 orthotopic bladder cancer F344 rat model was used. Rats were treated with consecutive intravesical instillations of CDC, BCG, the combination of CDC+BCG, or NaCl as control. Results CDC showed a dose-dependent antiproliferative effect on all human urothelial carcinoma cell lines tested and the rat AY-27 urothelial carcinoma cell line. Moreover, intravesical treatment with CDC and CDC+BCG results in a lower percentage of tumors (60% and 68%, respectively) compared to BCG (75%) or control (85%). This difference with placebo was not statistically significant (p=0.078 and 0.199, respectively). However, tumors present in the placebo and BCG-treated rats were generally of higher stage. Conclusions Cyclodextrin-curcumin complex showed an antiproliferative effect on human and rat urothelial carcinoma cell lines in vitro. In the aggressive orthotopic bladder cancer rat model, we observed a promising effect of CDC treatment and CDC in combination with BCG.