Panel Of Human Cancer Cells Research Articles

Highly luminescent metallacages featuring bispyridyl ligands functionalised with BODIPY for imaging in cancer cells

Recently, 3-dimensional supramolecular coordination complexes of the metallacage type have been shown to hold promise as drug delivery systems for different cytotoxic agents, including the anticancer drug cisplatin. However, so far only limited information is available on their uptake and sub-cellular localisation in cancer cells. With the aim of understanding the fate of metallacages in cells by fluorescence microscopy, three fluorescent Pd2L4 metallacages were designed and synthesised by self-assembly of two types of bispyridyl ligands (L), exo-functionalised with boron dipyrromethene (BODIPY) moieties, with Pd(II) ions. The cages show high quantum yields and are moderately stable in the presence of physiologically relevant concentration of glutathione. Furthermore, the cages are able to encapsulate the anticancer drug cisplatin, as demonstrated by NMR spectroscopy. Preliminary cytotoxicity studies in a small panel of human cancer cells showed that the metallacages are scarcely toxic in vitro. The marked fluorescence due to BODIPY allowed us to visualise the cages' uptake and sub-cellular localisation inside melanoma cells using fluorescence microscopy, highlighting uptake via active transport mechanisms and accumulation in cytoplasmic vesicles.

Abstract 2050: Cell penetrating peptide, ST101, disrupts ATF5 regulation of anti-apoptotic Bcl-2 family proteins, resulting in induction of cancer cell death in vitro and tumor growth inhibition/regression in vivo

Abstract Anti-apoptotic B cell lymphoma 2 (Bcl-2) family proteins are frequently overexpressed across a variety of tumors, resulting in tumor cell survival and resistance to therapy. Inhibition of the expression or activity of these survival factors is an attractive approach for cancer therapy. Activating transcription factor 5 (ATF5) regulates gene transcription of anti-apoptotic Bcl-2 family proteins in neural progenitor cells and a wide range of human cancer cells. Here, we describe ST101, a rationally designed, synthetic, D-amino acid, cell penetrating peptide therapeutic designed to disrupt the protein-protein interactions driving ATF5-regulated gene transcription. Exposure of HL60 promyelocytic leukemia cells and MCF7 breast adenocarcinoma cells to low micromolar concentration of ST101 resulted in a decrease in MCL-1, BCL-2 and BIRC5 (Survivin) mRNA expression at 4 and 24 hrs post exposure. Further, exposure to ST101 resulted in a dose-dependent loss of viability across a panel of human cancer cells, including MCF7, HL60, U251 glioblastoma, A375 melanoma, DU145 prostate cancer, and A549 lung adenocarcinoma, characterized by an increase in annexin V and PI staining by flow cytometry peaking 48 hrs post exposure, resulting in a median half maximal effective concentration (EC50) value of 4.0 micromolar. In contrast, normal human peripheral blood mononuclear cells and bone marrow mononuclear cells were resistant to ST101-mediated cell death, with >80 micromolar EC50 values. In mouse xenograft experiments, 25mg/kg ST101 administered three times per week for three weeks resulted in significant tumor regression in MCF7 and U251 subcutaneous tumors as well as tumor growth delay in HL60 subcutaneous tumors. Tumor growth remained significantly inhibited weeks after the last treatment in the MCF7 and U251 models. In summary, ST101 selectively kills cancer cell lines by decreasing BCL-2 family gene expression, resulting in significant reductions in tumor growth in mouse models. Taken together, these data validate ST101 as a potent peptide therapeutic candidate for a variety of solid tumor and hematologic malignancies. Citation Format: Jim A. Rotolo, Rick Ramirez, Mark Koester, Siok Leong, Lila Ghamsari, Gene Merutka, Barry J. Kappel. Cell penetrating peptide, ST101, disrupts ATF5 regulation of anti-apoptotic Bcl-2 family proteins, resulting in induction of cancer cell death in vitro and tumor growth inhibition/regression in vivo [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 2050.

Exploring the cytotoxic activity of new phenanthroline salicylaldimine Zn(II) complexes

Zinc(II) complexes bearing N-salicylideneglycinate (Sal-Gly) and 1,10-phenanthroline (phen) or phenanthroline derivatives [NN = 5-chloro-1,10-phenanthroline, 5-amine-1,10-phenanthroline (amphen), 4,7-diphenyl-1,10-phenanthroline (Bphen) and 5,6-epoxy-5,6-dihydro-1,10-phenanthroline] are synthesized. Complexes formulated as [Zn(NN)2(H2O)2]2+(NN = phen and amphen), are also prepared. The cytotoxicity of the compounds is evaluated towards a panel of human cancer cells: ovarian (A2780), breast (MCF7) and cervical (HeLa), as well as non-tumoral V79 fibroblasts. All compounds display higher cytotoxicity than cisplatin (IC50 = 22.5 ± 5.0 μM) towards ovarian cells, showing IC50values in the low micromolar range. Overall, all compounds show higher selectivity for the A2780 cells than for the non-tumoral cells and higher selectivity indexes (IC50(V79)/IC50(A2780) than cisplatin. [Zn(Sal-Gly)(NN)(H2O)] complexes induce caspase-dependent apoptosis in A2780 cells, except [Zn(Sal-Gly)(Bphen)(H2O)], one of the most cytotoxic of the series. The cellular uptake in the ovarian cells analyzed by Inductively Coupled Plasma mass spectrometry indicates different Zn distribution profiles. Transmission electronic microscopy shows mitochondria alterations and apoptotic features consistent with caspase activation; cells incubated with [Zn(Sal-Gly)(amphen)(H2O)] present additional nuclear membrane alterations in agreement with significant association with the nucleus. The increase of reactive oxygen species and lipid peroxidation forms could be related to apoptosis induction. [Zn(NN)2(H2O)2]2+complexes have high ability to bind DNA through intercalation/groove binding, and circular dichroism data suggests that the main type of species that interact with DNA is [Zn(NN)]2+. Studies varying the % of fetal bovine serum (1–15%) in cell media show that albumin binding decreases the complex activity, indicating that distinct speciation of Zn- and phen-containing species in cell media may affect the cytotoxicity.

Development of conformational antibodies targeting Cripto-1 with neutralizing effects in vitro

Human Cripto-1 (Cripto-1), the founding member of the EGF-CFC superfamily, is a key regulator of many processes during embryonic development and oncogenesis. Cripto-1 is barely present or even absent in normal adult tissues while it is aberrantly re-expressed in various tumors. Blockade of the CFC domain-mediated Cripto-1 functions is acknowledged as a promising therapeutic intervention point to inhibit the tumorigenic activity of the protein.In this work, we report the generation and characterization of murine monoclonal antibodies raised against the synthetic folded CFC [112−150] domain of the human protein. Through subtractive ELISA assays clones were screened for the ability to specifically recognize “hot spot” residues on the CFC domain, which are crucial for the interaction with Activin Type I receptor (ALK4) and GRP78. On selected antibodies, SPR and epitope mapping studies have confirmed their specificity and have revealed that recognition occurs only on a conformational epitope.Furthermore, FACS analyses have confirmed the ability of 1B4 antibody to recognize the membrane-anchored and soluble native Cripto-1 protein in a panel of human cancer cells. Finally, we have evaluated its functional effects through in vitro cellular signaling assays and cell cycle analysis. These findings suggest that the selected anti-CFC mAbs have the potential to neutralize the protein oncogenic activity and may be used as theranostic molecules suitable as tumor homing agents for Cripto-1-overexpressing cancer cells and tissues and to overcome drug-resistance in routine cancer therapies.

Contributory role of microRNAs in anti-cancer effects of small molecule inhibitor of telomerase (BIBR1532) on acute promyelocytic leukemia cell line

Telomerase-mediated immortalization and proliferation of tumor cells is a promising anti-cancer treatment strategy and development of potent telomerase inhibitors is believed to open new window of treatments in human malignancies. In the present study, we found that BIBR1532, a small molecule inhibitor of human telomerase, exerted cytotoxic effects on a panel of human cancer cells spanning from solid tumors to hematologic malignancies; however, as compared with solid tumors, leukemic cells were more sensitive to this inhibitor. This was independent of molecular status of p53 in the leukemic cells. The results of a miRNA PCR array revealed that BIBR1532-induced cytotoxic effects in NB4, the most sensitive cell line, was coupled with alteration in a substantial number of cancer-related miRNAs. Interestingly, most of these miRNAs were found to act as tumor suppressors with validated targets in cell cycle or nuclear factor (NF)-κB–mediated apoptosis. In accordance with a bioinformatics analysis, our experimental studies showed that BIBR1532-induced apoptosis is mediated, at least partly, by inhibition of NF-κB. Moreover, we found that the alteration in the expression of miRNAs was coupled with the alteration in the cell cycle progression. To sum up with, a straightforward interpretation of our results is that telomerase inhibition using BIBR1532 not only induced CDKN1A-mediated G1 arrest in NB4, but also resulted in a caspase-3-dependent apoptotic cell death mostly through suppression of NF-κB axis.

Gold(III) Pyridine-Benzimidazole Complexes as Aquaglyceroporin Inhibitors and Antiproliferative Agents

Gold compounds have been proven to be novel and versatile tools for biological applications, including as anticancer agents. Recently, we explored the potential of Au(III) complexes with bi-dentate N-donor ligands as inhibitors of the membrane water and glycerol channels aquaporins (AQPs), involved in different physiological and pathophysiological pathways. Here, eight new Au(III) complexes featuring a pyridine-benzimidazole scaffold have been synthesized and characterized via different methods. The stability of all the compounds in aqueous solution and their reactivity with glutathione have been investigated by UV–visible spectroscopy. The Au(III) compounds, tested for their AQPs inhibition properties in human Red Blood Cells (hRBC), are potent and selective inhibitors of AQP3. Furthermore, the compounds’ antiproliferative effects have been studied in a small panel of human cancer cells expressing AQP3. The complexes show only very moderate anticancer effects in vitro and are mostly active against the melanoma A375 cells, with marked expression of AQP3 at the level of the nuclear membrane. In general, the AQP3 inhibition properties of these complexes hold promises to develop them as chemical probes to study the function of this protein isoform in biological systems.

Relevance of Copper and Organic Cation Transporters in the Activity and Transport Mechanisms of an Anticancer Cyclometallated Gold(III) Compound in Comparison to Cisplatin.

The molecular mechanisms of toxicity and cellular transport of anticancer metallodrugs, including platinum-based agents, have not yet been fully elucidated. The aim of our study was to investigate the relevance of copper transporters (CTR1 and ATP7A/B), organic cation transporters (OCT2) and the multidrug and toxin extrusion proteins (MATE) in the intracellular accumulation of a novel organometallic cytotoxic Au(III) compound in cancer cells in comparison to cisplatin. Specifically, the synthesis and characterization of the gold complex [Au(pyb-H)(PPh2Ar)Cl]PF6 (PPh2Ar = 3-[4-(diphenylphosphino)phenyl]-7-methoxy-2H-chromen-2-one] (1), featuring a coumarin ligand endowed with “smart” fluorescence properties, have been achieved. Initially, the cytotoxic effects of both cisplatin and 1 were studied in a small panel of human cancer cells, and against a non-tumorigenic cell line in vitro. Thus, the human ovarian cancer cell line A2780 and its cisplatin resistant variant A2780cisR, were selected, being most sensitive to the treatment of the gold complex. Co-incubation of the metallodrugs with CuCl2 (a CTR1 substrate) increased the cytotoxic effects of both the Au(III) complex and cisplatin; while co-incubation with cimetidine (inhibitor of OCT2 and MATE) showed some effect only after 72 h incubation. ICP-MS (Inductively Coupled Plasma Mass Spectrometry) analysis of the cell extracts showed that co-incubation with CuCl2 increases Au and Cu accumulation in both cancer cell lines, in accordance with the enhanced antiproliferative effects. Conversely, for cisplatin, no increase in Pt content could be observed in both cell lines after co-incubation with either CuCl2 or cimetidine, excluding the involvement of CTR1, OCT2, and MATE in drug accumulation and overall anticancer effects. This result, together with the evidence for increased Cu content in A2780 cells after cisplatin co-treatment with CuCl2, suggests that copper accumulation is the reason for the observed enhanced anticancer effects in this cell line. Moreover, metal uptake studies in the same cell lines indicate that both 1 and cisplatin are not transported intracellularly by CTR1 and OCT2. Finally, preliminary fluorescence microscopy studies enabled the visualization of the sub-cellular distribution of the gold compound in A2780 cells, suggesting accumulation in specific cytosolic components/organelles.

Mixed-ligand Cu(II) hydrazone complexes designed to enhance anticancer activity

The ligand quantity, ligand type, and coordination geometry have important influences on the anticancer activity of metal-based complexes. On the basis of the structures of previously reported 1:1 Cu(II)/ligand complexes ([Cu(L1)Cl]·2H2O 1a, [Cu(L2)Cl]·H2O 2a, and [Cu(L2)NO3]·H2O 3a), we subsequently designed, developed, and characterized a series of corresponding 1:1:1 Cu(II)/ligand/co-ligand complexes ([Cu(L1)(Py)Cl]·H2O 1b, [Cu(L2)(Py)Cl] 2b, and [Cu(L2)(Py)NO3] 3b), where L1 = (E)-N'-(2-hydroxybenzylidene)acetohydrazide, L2 = (E)-N'-(2- hydroxybenzylidene)benzohydrazide, and Py = pyridine. All six Cu(II) complexes were assessed for their in vitro anticancer properties against a panel of human cancer cells, including cisplatin-resistant A549cisR cell lines. Interestingly, we observed that the 1:1:1 Cu/ligand/co-ligand mixed-ligand Cu(II) complexes exhibited higher anticancer activity than the corresponding 1:1 Cu(II)/ligand complexes. In particular, the 1:1:1 Cu(II)/ligand/co-ligand complex 3b displayed the greatest toxicity toward several cancer cells with better IC50 (1.12–3.77 μM) than cisplatin. Further mechanistic explorations showed that the 3b complex induced DNA damage, thus resulting in mitochondria-mediated apoptotic cell death. Furthermore, the 3b complex displayed pronounced cytostatic effects in the MCF-7 3D spheroid model.

PO-022 Benzamide class of HDACi differentially enhance sodium iodide symporter expression in breast cancer

IntroductionSodium iodide symporter (hNIS) expression is reported across all subtypes of breast cancer (BC) and is a potential endogenous gene target for radioiodine therapy [PlosOne8(1:e54055, 2012]. Successful clinical application however relies on very high expression of functional NIS in BC cells. We have been exploiting various transcriptional regulation approaches for NIS, of which use of HDAC inhibitors (HDACi) have shown promising amplification of endogenous NIS expression in BC [Scientific Reports6:19341, 2016]. Here, we show that differential induction of NIS expression in BC is possible using Benzamide class of HDACi (bHDACi) with minimal off-target effects on other cell types.Material and methodsThe effect of bHDACi, i.e. CI-994, Chidamide, MS-275 and AR-42, on NIS gene transcription was verified on a panel of human cancer cells by RTPCR. Functional in vitro assessment was done by non-radioactive iodine uptake. I131therapeutic efficacy was measured by clonogenic assay. Promoter binding transcription factor (TF) profiler array was done to identify novel TFs responsible for differential activation in BC cells. Further, in vivo therapeutic efficacy was monitored by bioluminescence imaging (BLI) of ZR-75–1-fl2.tdt orthotopic model.Results and discussionsUsing a non-toxic, low dose of bHDACi(s) treatment on a cancer cell panel, including breast, thyroid, lung, glioma, fibrosarcoma and ovarian, a significant induction of NIS expression (p<0.05) was observed only in BC cells. Observed differences of NIS expression was confirmed by protein expression and functional assessment on BC cells. Further, novel TFs (FOXA1, FOXOF2 etc.) were identified which show differential activation in BC after CI-994 treatment. Only in case of BC cells pre-treated with bHDACi(s), cell survival assay results showed enhanced cell death on exposure to I131. Further, AR-42 drug was tested in vivo which showed similar results. AR-42 pre-treated ZR-75–1 fl2tdt orthotopic tumour showed increased NIS expression. In comparison to untreated and only I131 treated group, a combination treatment of AR-42 with 1mCi I131, BLI imaging showed 20% and 30% signal drop on day1 and day 2 respectively indicating specific radio-ablation effect.ConclusionbHDACi(s) show differentially higher regulation of endogenous NIS with minimal effect on non-breast tissues and thus may potentially benefit NIS-based radioablation therapy trials in BC patients with low/moderate expression of NIS.

Enediynes bearing polyfluoroaryl sulfoxide as new antiproliferative agents with dual targeting of microtubules and DNA

A novel series of enediynes possessing pentafluorophenylsulfoxide have been developed. The innovative compounds possess antiproliferative activity against a broad panel of human cancer cells originating from breast, blood, lung, kidney, colon, prostate, pancreas or skin with IC50 ranging from 0.6 to 3.4 μM. The antiproliferative activity of enediynes in darkness is associated to their ability to compromise microtubule network. In addition, exposure to UV leads to double-stranded DNA cleavage caused by the newly synthesized molecules reducing further their IC50 in nanomolar range against human tumor cells, including chemo-resistant pancreatic cancer cells. Taken together, the examined data demonstrate that enediynes possessing pentafluorosulfoxide are promising molecules in the cancer therapy.

Loss of Uracil DNA Glycosylase Selectively Resensitizes p53-Mutant and -Deficient Cells to 5-FdU.

Thymidylate synthase (TS) inhibitors including fluoropyrimidines [e.g., 5-Fluorouracil (5-FU) and 5-Fluorodeoxyuridine (5-FdU, floxuridine)] and antifolates (e.g., pemetrexed) are widely used against solid tumors. Previously, we reported that shRNA-mediated knockdown (KD) of uracil DNA glycosylase (UDG) sensitized cancer cells to 5-FdU. Because p53 has also been shown as a critical determinant of the sensitivity to TS inhibitors, we further interrogated 5-FdU cytotoxicity after UDG depletion with regard to p53 status. By analyzing a panel of human cancer cells with known p53 status, it was determined that p53-mutated or -deficient cells are highly resistant to 5-FdU. UDG depletion resensitizes 5-FdU in p53-mutant and -deficient cells, whereas p53 wild-type (WT) cells are not affected under similar conditions. Utilizing paired HCT116 p53 WT and p53 knockout (KO) cells, it was shown that loss of p53 improves cell survival after 5-FdU, and UDG depletion only significantly sensitizes p53 KO cells. This sensitization can also be recapitulated by UDG depletion in cells with p53 KD by shRNAs. In addition, sensitization is also observed with pemetrexed in p53 KO cells, but not with 5-FU, most likely due to RNA incorporation. Importantly, in p53 WT cells, the apoptosis pathway induced by 5-FdU is activated independent of UDG status. However, in p53 KO cells, apoptosis is compromised in UDG-expressing cells, but dramatically elevated in UDG-depleted cells. Collectively, these results provide evidence that loss of UDG catalyzes significant cell death signals only in cancer cells mutant or deficient in p53.Implications: This study reveals that UDG depletion restores sensitivity to TS inhibitors and has chemotherapeutic potential in the context of mutant or deficient p53. Mol Cancer Res; 16(2); 212-21. ©2017 AACR.

Triple action Pt(iv) derivatives of cisplatin: a new class of potent anticancer agents that overcome resistance.

A series of triple action Pt(iv) prodrugs was designed to test the hypothesis that multi-action compounds, where each bioactive moiety intervenes in several cellular processes, might be more effective than a single agent at killing cancer cells. In particular, "triple action" Pt(iv) derivatives of cisplatin, where the axial ligands are inhibitors of cyclooxygenase (COXi), histone deacetylase (HDACi) or pyruvate dehydrogenase kinase (PDKi) were developed. All compounds, ctc-[Pt(NH3)2(COXi)(PDKi)Cl2], ctc-[Pt(NH3)2(COXi)(HDACi)Cl2] and ctc-[Pt(NH3)2(HDACi)(PDKi)Cl2], where COXi = aspirin or ibuprofen, PDKi = dichloroacetate and HDACi = valproate or phenylbutyrate, were significantly more cytotoxic than cisplatin against all cell lines of an in-house panel of human cancer cells. They were particularly effective against thyroid and pancreatic cancer cells in monolayer cytotoxicity tests. Remarkably, in 3D spheroid cancer cell cultures, some triple action compounds showed an antitumor potency up to 50-fold higher than cisplatin against a KRAS mutated pancreatic cancer cell line (PSN-1 cells). Standard biochemical assays classically employed to explore structure activity relationships of platinum drugs, such as cellular uptake and binding to potential biological targets (DNA, HDAC, mitochondria, and COX), do not provide linear correlations with the overall cytotoxicity data. We observed a preferential induction of ROS production and of an anti-mitochondrial effect in cancer cells compared to rapidly dividing non-cancerous cells. Thus, we propose that these new triple action Pt(iv) derivatives of cisplatin are a novel and interesting class of potent and selective cytotoxic agents.

Alkaloids from Narcissus poeticus cv. Pink Parasol of various structural types and their biological activity.

Fifteen Amaryllidaceae alkaloids (1-15) of various structural types were isolated by standard chromatographic methods from fresh bulbs of Narcissus poeticus cv. Pink Parasol. The chemical structures were elucidated by MS, and 1D and 2D NMR spectroscopic analyses, and by comparison with literature data. Narcipavline (5) and narcikachnine (6) are reported here for the first time. In their structure are combined two basic structural types of Amaryllidaceae alkaloids (galanthamine- and galanthindole-structural types), which represent a new structural type of these compounds. Alkaloids isolated in sufficient amounts were evaluated for their human erythrocytic acetylcholinesterase, and human serum butyrylcholinesterase (HuBuChE) inhibition activity using Ellman's method. Z-Gly-Pro-p-nitroanilide was used as substrate in the prolyl oligopeptidase (POP) assay. Untested alkaloids were also screened for their cytotoxic activity against a small panel of human cancer cells, which spanned cell lines from different tissue types. In parallel, MRC-5 human fibroblasts were employed to determine overall toxicity against noncancerous cells. Some compounds were evaluated for their antiprotozoal activity. The newly isolated alkaloid narcipavline (5) showed interesting HuBuChE inhibition activity (IC50=24.4±1.2µM), and norlycoramine (11) demonstrated promising POP inhibition (IC50=0.21±0.01mM).

MRPL33 and its splicing regulator hnRNPK are required for mitochondria function and implicated in tumor progression.

MRPL33 gene encodes a large mitoribosomal subunit protein, which may be involved in mitochondrial translation. Although two splice variants of MRPL33 have been described, its splicing regulation remains elusive. Here we observed that inclusion of alternative exon 3 was greatly promoted in a panel of human cancer cells. Depletion of the exon 3-containing long isoform of MRPL33 (MRPL33-L) led to impaired proliferation and increased apoptosis in cancer cell lines and in a xenograft model. MRPL33-L knockdown could also induce mitochondrial dysfunction including increased accumulation of reactive oxygen species, decreased ATP production and 16 S rRNA levels. We further showed that alternative splicing of MRPL33-L pre-mRNA is regulated by hnRNPK and that knocking down hnRNPK could phenocopy MRPL33-L depletion. More importantly, overexpression of MRPL33-L could increase tumorigenic potential of hnRNPK-depleted cancer cells, likely indicating that hnRNPK mediates tumorigenesis through splicing regulation of MRPL33 pre-mRNA. Finally, we found that inclusion of MRPL33 exon 3 was promoted in human colorectal cancer tissues and this was correlated with hnRNPK levels. In summary, our findings underscore the biological significance of MRPL33-L and hnRNPK in the tumor formation and identifies hnRNPK as a critical splicing regulator of MRPL33 pre-mRNA in cancer cells.

Cladosporol A triggers apoptosis sensitivity by ROS-mediated autophagic flux in human breast cancer cells

BackgroundEndophytes have proven to be an invaluable resource of chemically diverse secondary metabolites that act as excellent lead compounds for anticancer drug discovery. Here we report the promising cytotoxic effects of Cladosporol A (HPLC purified >98%) isolated from endophytic fungus Cladosporium cladosporioides collected from Datura innoxia. Cladosporol A was subjected to in vitro cytotoxicity assay against NCI60 panel of human cancer cells using MTT assay. We further investigated the molecular mechanism(s) of Cladosporol A induced cell death in human breast (MCF-7) cancer cells. Mechanistically early events of cell death were studied using DAPI, Annexin V-FITC staining assay. Furthermore, immunofluorescence studies were carried to see the involvement of intrinsic pathway leading to mitochondrial dysfunction, cytochrome c release, Bax/Bcl-2 regulation and flowcytometrically measured membrane potential loss of mitochondria in human breast (MCF-7) cancer cells after Cladosporol A treatment. The interplay between apoptosis and autophagy was studied by microtubule dynamics, expression of pro-apoptotic protein p21 and autophagic markers monodansylcadaverine staining and LC3b expression.ResultsAmong NCI60 human cancer cell line panel Cladosporol A showed least IC50 value against human breast (MCF-7) cancer cells. The early events of apoptosis were characterized by phosphatidylserine exposure. It disrupts microtubule dynamics and also induces expression of pro-apoptotic protein p21. Moreover treatment of Cladosporol A significantly induced MMP loss, release of cytochrome c, Bcl-2 down regulation, Bax upregulation as well as increased monodansylcadaverine (MDC) staining and leads to LC3-I to LC3-II conversion.ConclusionOur experimental data suggests that Cladosporol A depolymerize microtubules, sensitize programmed cell death via ROS mediated autophagic flux leading to mitophagic cell death.Graphical abstractThe proposed mechanism of Cladosporol A -triggered apoptotic as well as autophagic death of human breast cancer (MCF-7) cells. The figure shows that Cladosporol A induced apoptosis through ROS mediated mitochondrial pathway and increased p21 protein expression in MCF-7 cells in vitro.

Cytotoxic Essential Oils from Eryngium campestre and Eryngium amethystinum (Apiaceae) Growing in Central Italy.

Eryngium campestre and E. amethystinum are thorny herbs belonging to the Apiaceae family and spontaneously growing in stony pastures and dry meadows, preferentially on calcareous substrates. In the Mediterranean countries, these plants have been used as a food or traditional remedies to treat various ailments. In the present work, we have analyzed the chemical composition of the essential oils distilled from the aerial parts by GC-FID and GC/MS, and evaluated their cytotoxic effects on a panel of human cancer cells, namely A375 (human malignant melanoma), MDA-MB 231 cells (human breast adenocarcinoma), and HCT116 cells (human colon carcinoma), by the MTT assay. Furthermore, the Eryngium essential oils were evaluated for antioxidant and acetylcholinesterase (AChE) activities. The two essential oils were rich in sesquiterpene hydrocarbons, with germacrene D as the major compound, accompanied by allo-aromadendrene, β-elemene, spathulenol, and ledol. They turned out to be highly cytotoxic on the tumor cells, with IC50 values (1.65 - 5.32 and 1.57 - 2.99 μg/ml for E. amethystinum and E. campestre, respectively) comparable or close to those of the anticancer drug cisplatin. The E. amethystinum essential oil exhibited a moderate antioxidant activity, whereas that of E. campestre a weak AChE inhibition.

Structure-Activity Relationship of Niclosamide Derivatives.

Cancer is a leading cause of death. Hence, this study aimed at the optimization of niclosamide derivatives for the development of new potential anticancer agents. Niclosamide derivatives were synthesized and tested against a panel of human cancer cells: MDA and MCF7 breast cancer cells, PC3 and DU-145 prostate cancer cells, Hela cervical cancer cells, and HL-60 acute promyelocytic leukemia cells. They were also tested in nuclear factor-ĸappa B (NFĸB), V-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS), and mitochondria transmembrane potential (MTP) assays. N-(3,5-Bis(trifluoromethyl)phenyl)-5-chloro-2-hydroxybenzamide exhibited the most significant cytotoxicity against HL-60 cells, while 5-chloro-N-(2-chlorophenyl)-2-hydroxybenzamide was the most active in the NFĸB assay and 5-chloro-N-(3,5-difluorophenyl)-2-hydroxybenzamide in the MTP assay. 5-chloro-N-(2-chloro-4-(trifluoromethyl) phenyl)-2-hydroxybenzamide and 5-chloro-2-hydroxy-N-(4-hydroxyphenyl)benzamide inhibited both HL-60 cell proliferation and NFĸB. In-depth study of the most promising compounds is highly encouraged to further develop into potential anticancer agents those derivatives found to be significantly active.

Identification of novel 1-indolyl acetate-5-nitroimidazole derivatives of combretastatin A-4 as potential tubulin polymerization inhibitors

Microtubules are essential for the mitotic division of cells and have become an attractive target for anti-tumour drugs due to the increased incidence of cancer and significant mitosis rate of tumour cells. In this study, a total of six indole 1-position modified 1-indolyl acetate-5-nitroimidazole derivatives were designed, synthesized, and evaluated for their ability to inhibit tubulin polymerization caused by binding to the colchicine-binding site of tubulin. Among them, compound 3 displayed the best ability to inhibit tubulin polymerization; it also exhibited better anti-proliferative activities than colchicine against a panel of human cancer cells (with IC50 values ranging from 15 to 40nM), especially HeLa cells (with IC50 values of 15nM), based on the cellular cytotoxicity assay results. Moreover, cellular mechanism studies indicated that compound 3 could induce G2/M phase arrest and apoptosis of HeLa and MCF-7 cells, which were associated with alterations in the expression of cell cycle-checkpoint related proteins (Cyclin B1, Cdc2, and P21) and a reduction in the mitochondrial membrane potential as well as alterations in the levels of apoptosis-related proteins (PARP, Caspase 9, Bcl-2, and Bax) of these cells, respectively. Importantly, in vivo studies further revealed that compound 3 could dramatically suppress HeLa cell xenograft tumour growth compared with vehicle and CA-4 phosphate (CA-4P), and no signs of toxicity were observed in these mice. Collectively, these in vitro and in vivo results indicated that compound 3 might be a promising lead compound for further development as a potential anti-cancer drug.

Phytochemical investigations and antiproliferative secondary metabolites from Thymus alternans growing in Slovakia

Context: Thymus alternans Klokov (Lamiaceae) is a neglected species of the genus Thymus (Sect. Serpyllum) endemic to Carpathian area, where it is used as a flavouring agent and for medicinal purposes. Objective: The aim of the work was to identify antiproliferative constituents from the flowering aerial parts of this plant. Materials and methods: Thymus alternans extracts were analyzed by HPLC-MSn and subjected to extensive chromatographic separations. The isolated compounds (phenolics and triterpenes) were structurally elucidated by MS and 1D and 2D NMR experiments. Essential oil (EO) composition was determined by GC-FID and GC-MS. Six purified triterpenes and EO were assayed for in vitro antiproliferative activity against a panel of human cancer cells, namely, breast (MDA-MB 231), colon (HCT-15 and HCT116), lung (U1810), pancreatic (BxPC3), melanoma (A375) and cervical carcinoma (A431) cells. Results: The structures of the isolated compounds were achieved on the basis of H-NMR and MS experiments. Luteolin-4′-O-β-d-glucopyranoside (P1), chrysoeriol-7-O-β-d-glucopyranoside (P2), chrysoeriol-5-O-β-d-glucopyranoside (P3), apigenin-7-O-β-d-glucopyranoside (P4), rosmarinic acid (P5), rosmarinic acid-3′-O-β-d-glucopyranoside (P6), caffeic acid-3-O-β-d-glucopyranoside (P7), 3α-hydroxy-urs-12,15-diene (T1), α-amyrin (T2), β-amyrin (T3), isoursenol (T4), epitaraxerol (T5), and oleanolic acid (T6). GC-MS analysis revealed that the EO of T. alternans was devoid of phenols and belonged to the nerolidol-chemotype, that is typical of the Sect. Serpyllum. The six purified triterpenes (T1-T6) were active with IC50 ranging from 0.5 to 5 μM being comparable or better than those of reference compounds betulinic acid and cisplatin. The EO exhibited significant effects on A375, MDA-MB 231 and HCT116 cell lines with IC50 in the range of 5–8 μg/mL. Conclusion: The reported results suggest that T. alternans can be considered as a good source of phytoconstituents with possible importance in the pharmaceutical field.

A map of mobile DNA insertions in the NCI-60 human cancer cell panel.

BackgroundThe National Cancer Institute-60 (NCI-60) cell lines are among the most widely used models of human cancer. They provide a platform to integrate DNA sequence information, epigenetic data, RNA and protein expression, and pharmacologic susceptibilities in studies of cancer cell biology. Genome-wide studies of the complete panel have included exome sequencing, karyotyping, and copy number analyses but have not targeted repetitive sequences. Interspersed repeats derived from mobile DNAs are a significant source of heritable genetic variation, and insertions of active elements can occur somatically in malignancy.MethodWe used Transposon Insertion Profiling by microarray (TIP-chip) to map Long INterspersed Element-1 (LINE-1, L1) and Alu Short INterspersed Element (SINE) insertions in cancer genes in NCI-60 cells. We focused this discovery effort on annotated Cancer Gene Index loci.ResultsWe catalogued a total of 749 and 2,100 loci corresponding to candidate LINE-1 and Alu insertion sites, respectively. As expected, these numbers encompass previously known insertions, polymorphisms shared in unrelated tumor cell lines, as well as unique, potentially tumor-specific insertions. We also conducted association analyses relating individual insertions to a variety of cellular phenotypes.ConclusionsThese data provide a resource for investigators with interests in specific cancer gene loci or mobile element insertion effects more broadly. Our data underscore that significant genetic variation in cancer genomes is owed to LINE-1 and Alu retrotransposons. Our findings also indicate that as large numbers of cancer genomes become available, it will be possible to associate individual transposable element insertion variants with molecular and phenotypic features of these malignancies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13100-016-0078-4) contains supplementary material, which is available to authorized users.