A2780cis Cells Research Articles

DNA interaction of a fluorescent, cytotoxic pyridinimino platinum(II) complex

New pyridinimino complexes of platinum(II) [PtCl2(N^N-R)] (N^N = 2-pyridylmethanimino, R = -(CH2)2O(CH2)2OH, -(CH)2O(CH2)2OCH2Pyr), Pyr = pyren-1-yl) have been prepared. They are characterized by a dioxygenated alkyl side chain and, in one case, by a fluorescent terminal 1-pyrenyl residue. The complexes were characterized by elemental analysis, IR, 1H–, 13C–and 195Pt NMR spectroscopies. For [PtCl2(N^N-(CH2)2O(CH2)2OH] the molecular structure was determined by single crystal X-ray diffraction. The complexes are soluble and stable in DMSO/H2O (80/20, v/v). The pyrenyl terminated compound was tested as antiproliferative agent against selected human cancer cell lines. Comparable cytotoxic effect was obtained on human ovarian carcinoma A-2780 and A-2780cis cells, thus suggesting a certain ability to circumvent cisplatin resistance. The interaction of this complex with DNA was investigated by linear flow dichroism and by spectrophotometric (absorbance and fluorescence) titrations. Both techniques enlightened the presence of a complex mode of interaction with DNA, involving both groove binding and intercalation.

Synthesis of New Cu Complex Based on Natural 5Z,9Z-Eicosadienoic Acid: Effective Topoisomerase I Inhibitor and Cytotoxin against the Cisplatin-Resistant Cell Line.

The complex (bipy)2Cu(5,9-eicd) was prepared by the reaction of Cu(OAc)2 with 5Z,9Z-eicosadienoic acid and 2,2′-bipyridine in methanol. The new copper complex showed high antitumor activity in vitro toward A2780cis, A2780, Hek293, K562, HL60, Jurkat, and U937 cell lines and efficiently inhibited human topoisomerase I. Using flow cytofluorometry, (bipy)2Cu(5,9-eicd) was studied for the effect on the cell cycle and apoptosis-inducing activity in tumor cells.

Abstract 715: Mechanism-based evaluation of apoptosis in live tumor cells by triple co-fluorescence staining: Testing algorithmic effectiveness of targeted drugs

Abstract Background: Suppression of apoptosis, a morphologically distinct form of cell death is a trait commonly shared by tumor cells which provide them survival advantages and enables them to evolve to drug-resistant states, higher stages, and/or metastasize. Recently we have reported a procedure of Triple co-Fluorescence Staining (FTS) to visualize apoptosis following chemotherapeutic and targeted anti-cancer drugs in live tumor cells (De et al., 2018). Aim: Using triple-co-fluorescence to simultaneously identify 3 crucial mechanisms of apoptosis, (1) enzyme activity of executioner-caspase3, (2) caspase-dependent cell surface exposure of phosphatidylserine and (3) functional mitochondria, we studied apoptosis following combinations of (a) paclitaxel plus BKM120, (b) trametinib plus paclitaxel or CDK4/6 inhibitor in ovarian models, and (c) p110beta inhibitor plus BNM673 in TNBC models. Methods: Ovarian cell lines OVK18, A2780Cis, and SUM149 (TNBC) were used for the study. For the standardization of MitoViewBlue (B) staining, a final concentration of 50 nM was used. Treated live cells on cover-slips were incubated under cultured conditions in the dark for 15-30 minutes with the staining cocktail containing MitoViewBlue (B), NucView® 488 (G) and CF®594 Annexin V (R) in the 1X binding buffer. At the end of the incubation period, pictures were taken (Olympus DP72 digital camera) within a span of 10 minutes. The FTS was corroborated with (1) AnnexinV by flow cytometry, (2) live/dead cell assay, (3) WB expression of apoptotic markers, and (4) mitochondrial depolarization. Results: Using a standardized protocol for FTS we tested apoptosis in ovarian and TNBC models following the combination of chemotherapeutic and targeted anti-cancer drugs. Using B, we stained active mitochondria while using G we stained active caspase3 and R was used to localize plasma membrane asymmetry, flipped phosphatidylserine, a characteristic feature of apoptotic cells. A merged TFS image of live and non-treated (NT) OVK18, A2780Cis and SUM149 cells showed functional mitochondrial stained with bright blue which corroborated with TMRE-based mitochondrial potential. Fewer cells showed membrane fluorescence in red for R and even fewer cells with green fluorescence for G which stained active caspase3. R and G were mutually exclusive. Paclitaxel-treated A2780 cells were significantly devoid of B stains. Membrane blebbing and annexin-V stained cell membrane was identified in cells either with or without G stains but definitely without B stains which indicated the sequence of events of apoptotic in cells. The mutual exclusivity property of B cell to R and/or G cells are preserved as observed with the help of transmission overlay images. Conclusion: TFS can be used to evaluate mechanism-based apoptosis following chemotherapeutic and targeted anti-cancer drugs in live tumor cells. Citation Format: Nandini Dey, Jennifer C. Aske, Casey Williams, Pradip K. De, Brian Leyland-Jones. Mechanism-based evaluation of apoptosis in live tumor cells by triple co-fluorescence staining: Testing algorithmic effectiveness of targeted drugs [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 715.

Adenosine Analogues as Opposite Modulators of the Cisplatin Resistance of Ovarian Cancer Cells.

Adenosine released by cancer cells in high amounts in the tumour microenvironment is one of the main immunosuppressive agents responsible for the escape of cancer cells from immunological control. Blocking adenosine receptors with adenosine analogues and restoring immune cell activity is one of the methods considered to increase the effectiveness of anticancer therapy. However, their direct effects on cancer cell biology remain unclear. Here, we determined the effect of adenosine analogues on the response of cisplatinsensitive and cisplatin-resistant ovarian cancer cells to cisplatin treatment. The effects of PSB 36, DPCPX, SCH58261, ZM 241385, PSB603 and PSB 36 on cisplatin cytotoxicity were determined against A2780 and A2780cis cell lines. Quantification of the synergism/ antagonism of the compounds cytotoxicity was performed and their effects on the cell cycle, apoptosis/necrosis events and cisplatin incorporation in cancer cells were determined. PSB 36, an A1 receptor antagonist, sensitized cisplatin-resistant ovarian cancer cells to cisplatin from low to high micromolar concentrations. In contrast to PSB 36, the A2AR antagonist ZM 241385 had the opposite effect and reduced the influence of cisplatin on cancer cells, increasing their resistance to cisplatin cytotoxicity, decreasing cisplatin uptake, inhibiting cisplatin-induced cell cycle arrest, and partly restoring mitochondrial and plasma membrane potentials that were disturbed by cisplatin. Adenosine analogues can modulate considerable sensitivity to cisplatin of ovarian cancer cells resistant to cisplatin. The possible direct beneficial or adverse effects of adenosine analogues on cancer cell biology should be considered in the context of supportive chemotherapy for ovarian cancer.

Promotion of Cell Death in Cisplatin-Resistant Ovarian Cancer Cells through KDM1B-DCLRE1B Modulation.

Ovarian cancer is the gynecological malignancy with the poorest prognosis, in part due to its high incidence of recurrence. Platinum agents are widely used as a first-line treatment against ovarian cancer. Recurrent tumors, however, frequently demonstrate acquired chemo-resistance to platinum agent toxicity. To improve chemo-sensitivity, combination chemotherapy regimens have been investigated. This study examined anti-tumor effects and molecular mechanisms of cytotoxicity of Oldenlandia diffusa (OD) extracts on ovarian cancer cells, in particular, cells resistant to cisplatin. Six ovarian cancer cells including A2780 and cisplatin-resistant A2780 (A2780cis) as representative cell models were used. OD was extracted with water (WOD) or 50% methanol (MOD). MOD significantly induced cell death in both cisplatin-sensitive cells and cisplatin-resistant cells. The combination treatment of MOD with cisplatin reduced viability in A2780cis cells more effectively than treatment with cisplatin alone. MOD in A2780cis cells resulted in downregulation of the epigenetic modulator KDM1B and the DNA repair gene DCLRE1B. Transcriptional suppression of KDM1B and DCLRE1B induced cisplatin sensitivity. Knockdown of KDM1B led to downregulation of DCLRE1B expression, suggesting that DCLRE1B was a KDM1B downstream target. Taken together, OD extract effectively promoted cell death in cisplatin-resistant ovarian cancer cells under cisplatin treatment through modulating KDM1B and DCLRE1B.

The effect of sodium butyrate and cisplatin on expression of EMT markers.

Histone modifications play a key role in the epigenetic regulation of gene transcription in cancer cells. Histone acetylations are regulated by two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs are increased in ovarian carcinomas and they are involved in carcinogenesis and resistance to chemotherapeutic agents. In our study we investigated anticancer effect of HDAC inhibitor sodium butyrate (NaBu) on cisplatin-sensitive and cisplatin-resistant ovarian cell lines A2780 and A2780cis. A2780 and A2780cis were treated with NaBu alone or in combination with cisplatin (CP). NaBu inhibited the growth of both cell lines and enhanced cytotoxic effect of CP. Exposure to NaBu for 24 h induced cell cycle arrest. The expressions of EMT-related genes and proteins were further investigated by qPCR and western blot analysis. Loss of E-cadherin has been shown to be crucial in ovarian cancer development. We found that NaBu dramatically induce expression of E-cadherin gene (CDH1) and protein levels in A2780 and A2780cis. We investigated correlation between transcription and methylation of CDH1gene. Methylation level analysis in 32 CpG sites in CDH1 gene (promoter/exon1 regions) was performed using bisulfite NGS (Next Generation Sequencing). We found that cisplatin-resistant cell line A2780cis cells differ from their cisplatin-sensitive counterparts in the CDH1 methylation. Methylation in A2780cis cells is elevated compared to A2780. However, NaBu-induced expression of CDH1 was not accompanied by CDH1 demethylation. NaBu treatment induced changes in expression of EMT-related genes and proteins. Interestingly E-cadherin zinc finger transcriptional repressor SNAIL1 was upregulated in both cell lines. Mesenchymal marker vimentin was downregulated. Matrix metalloproteases (MMPs) are necessary for pericellular proteolysis and facilitate migration and invasion of tumour cells. NaBu induced mRNA expression of MMPs, mild changes in activities of gelatinases MMP2 and MMP9 were detected. Our data demonstrate that NaBu sensitizes cisplatin-resistant ovarian cancer cells, re-established E-cadherin expression, but it was not able to reverse the EMT phenotype completely.

Platinum(iv) dihydroxido diazido N-(heterocyclic)imine complexes are potently photocytotoxic when irradiated with visible light.

A series of trans-di-(N-heterocyclic)imine dihydroxido diazido PtIV complexes of the form trans,trans,trans-[Pt(N3)2(OH)2(L1)(L2)] where L = pyridine, 2-picoline, 3-picoline, 4-picoline, thiazole and 1-methylimidazole have been synthesised and characterised, and their photochemical and photobiological activity evaluated. Notably, complexes 19 (L1 = py, L2 = 3-pic) and 26 (L1 = L2 = 4-pic) were potently phototoxic following irradiation with visible light (420 nm), with IC50 values of 4.0 μM and 2.1 μM respectively (A2780 cancer cell line), demonstrating greater potency than the previously reported complex 1 (L1 = L2 = py; 6.7 μM); whilst also being minimally toxic in the absence of irradiation. Complexes with mixed N-(heterocyclic)imine ligands 19 and 20 (L1 = py, L2 = 4-pic) were particularly photocytotoxic towards cisplatin resistant (A2780cis) cell lines. Complex 18 (L1 = py, L2 = 2-pic) was comparatively less photocytotoxic (IC50 value 14.5 μM) than the other complexes, despite demonstrating the greatest absorbance at the irradiation wavelength and the fastest half-life for loss of the N3 → Pt LMCT transition upon irradiation (λ irr = 463 nm) in aqueous solution. Complex 29 (X1 = X2 = thiazole) although potently phototoxic (2.4 μM), was also toxic towards cells in the absence of irradiation.

Small Molecules Identified from a Quantitative Drug Combinational Screen Resensitize Cisplatin's Response in Drug-Resistant Ovarian Cancer Cells

Drug resistance to chemotherapy occurs in many ovarian cancer patients resulting in failure of treatment. Exploration of drug resistance mechanisms and identification of new therapeutics that overcome the drug resistance can improve patient prognosis. Following a quantitative combination screen of 6060 approved drugs and bioactive compounds in a cisplatin-resistant A2780-cis ovarian cancer cell line, 38 active compounds with IC50s under 1 μM suppressed the growth of cisplatin-resistant ovarian cancer cells. Among these confirmed compounds, CUDC-101, OSU-03012, oligomycin A, VE-821, or Torin2 in a combination with cisplatin restored cisplatin's apoptotic response in the A2780-cis cells, while SR-3306, GSK-923295, SNX-5422, AT-13387, and PF-05212384 directly suppressed the growth of A2780-cis cells. One of the mechanisms for overcoming cisplatin resistance in these cells is mediated by the inhibition of epidermal growth factor receptor (EGFR), though not all the EGFR inhibitors are equally active. The increased levels of total EGFR and phosphorylated-EGFR (p-EGFR) in the A2780-cis cells were reduced after the combined treatment of cisplatin with EGFR inhibitors. In addition, a knockdown of EGFR mRNA reduced cisplatin resistance in the A2780-cis cells. Therefore, the top active compounds identified in this work can be studied further as potential treatments for cisplatin-resistant ovarian cancer. The quantitative combinational screening approach is a useful method for identifying effective compounds and drug combinations against drug-resistant cancer cells.

Glutathione-Scavenging Poly(disulfide amide) Nanoparticles for the Effective Delivery of Pt(IV) Prodrugs and Reversal of Cisplatin Resistance.

Despite the broad antitumor spectrum of cisplatin, its therapeutic efficacy in cancer treatment is compromised by the development of drug resistance in tumor cells and systemic side effects. A close correlation has been drawn between cisplatin resistance in tumor cells and increased levels of intracellular thiol-containing species, especially glutathione (GSH). The construction of a unique nanoparticle (NP) platform composed of poly(disulfide amide) polymers with a high disulfide density for the effective delivery of Pt(IV) prodrugs capable of reversing cisplatin resistance through the disulfide-group-based GSH-scavenging process, as described herein, is a promising route by which to overcome limitations associated with tumor resistance. Following systematic screening, the optimized NPs (referred to as CP5 NPs) showed a small particle size (76.2 nm), high loading of Pt(IV) prodrugs (15.50% Pt), a sharp response to GSH, the rapid release of platinum (Pt) ions, and notable apoptosis of cisplatin-resistant A2780cis cells. CP5 NPs also exhibited long blood circulation and high tumor accumulation after intravenous injection. Moreover, in vivo efficacy and safety results showed that CP5 NPs effectively inhibited the growth of cisplatin-resistant xenograft tumors with an inhibition rate of 83.32% while alleviating serious side effects associated with cisplatin. The GSH-scavenging nanoplatform is therefore a promising route by which to enhance the therapeutic index of Pt drugs used currently in cancer treatment.

Back Cover: Optimized two‐dimensional gel electrophoresis in an alkaline pH range improves the identification of intracellular CFDA‐cisplatin‐protein adducts in ovarian cancer cells

DOI: 10.1002/elps.201700377 The cover picture shows the visualization of CFDA-cisplatin-protein adducts through 2DE. Human ovarian carcinoma cell lines A2780 and A2780cis cells were treated with CFDA-cisplatin and separated by 2DE. The use of fluorescent CFDA-cisplatin enables the visualization of CFDA-cisplatin-protein adducts. For the image analysis the fluorescence scans from CFDA-cisplatin-protein adducts and the Coomassie staining were fused into one image. The identification of nine previously unknown intracellular binding partners of CFDA-cisplatin by mass spectrometry provides new insights into the cellular processing of cisplatin.

Highly Emissive Self-Assembled BODIPY-Platinum Supramolecular Triangles.

Light-emitting supramolecular coordination complexes (SCCs) have been widely studied for applications in the chemical and biological sciences. Herein, we report the coordination-driven self-assembly of two highly emissive platinum(II) supramolecular triangles (1 and 2) containing BODIPY-based bridging ligands. The metallacycles exhibit favorable anticancer activities against HeLa cells (IC50 of 6.41 and 2.11 μM). The characteristic ∼570 nm fluorescence of the boron dipyrromethene (BODIPY) moieties in the metallacycles permits their intracellular visualization using confocal microscopy. Additionally, the BODIPY fluorophore is an excellent photodynamic agent, making the metallacycles as ideal therapeutics for photodynamic therapy (PDT) and chemotherapy. In vitro studies demonstrate that the combination indexes against HeLa cells are 0.56 and 0.48 for 1 and 2, respectively, confirming their synergistic anticancer effect. More importantly, these SCCs also exhibit superior anticancer efficacy toward cisplatin-resistant A2780cis cell line by combining PDT and chemotherapy, showing promise in overcoming drug resistance. This study exploits a multicomponent approach to self-assembled metallacages that enables design of effective theranostic agents wherein the platinum acceptors are toxic chemotherapeutics and the BODIPY donors are imaging probes and photosensitizers. Since each piece may be independently tuned, i.e., Pt(II) polypyridyl fragment swapped for Pt(II) phosphine, the activity may be optimized without a total redesign of the system.

Cisplatin-induced ERK1/2 activity promotes G1 to S phase progression which leads to chemoresistance of ovarian cancer cells.

The link between ERK1/2 activity and cisplatin cytotoxicity, in association with the cell cycle, in ovarian cancer cell lines resistant (A2780cis; SK-OV-3) and sensitive (A2780) to cisplatin was determined. We observed that cisplatin, at a low concentration enhanced the activation of ERK1/2 in A2780 cells and increased their accumulation in the S phase, resulting in low cytotoxicity. A high concentration of drug induced dephosphorylation and degradation of ERK1/2 and was extremely toxic, accumulating most of to these cells in the sub-G1 phase. The PD98059, pharmacological inhibitor of ERK1/2 activation, increased the cytotoxicity of cisplatin applied at a low concentration to A2780 cells (decreased ERK1/2 activity), causing shift of cell accumulation from the S to G1 phase. Surprisingly, PD98059 enhanced cell viability when a chemotherapeutic was used at high concentration, intensifying phosphorylation level of ERK1/2 and reversing cell cycle arrest in sub-G1 to promote the G1 and S phases. A2780cis cells demonstrated resistance to cisplatin with high ERK1/2 activity and accumulation of cells in the G1 and S phases. PD98059 sensitized resistant cells to drug toxicity during the first 24 hours of treatment, with blocked ERK1/2 phosphorylation and prevented progression from the G1 to S phase. SK-OV-3 resistant cells characterized with extremely high basal phosphorylation of ERK1/2, which wasn't changed after exposure to cisplatin. Administration of PD98059 didn't change the cytotoxicity of cisplatin in these cells. In conclusion, ERK1/2, activated by cisplatin, participates in the cell cycle progression from the G1 to S phase, enhancing cells’ survival and drug resistance.

Triphenyltin derivatives of sulfanylcarboxylic esters

The reaction of 3-(aryl)-2-sulfanylpropenoic acids [H2xspa; x: p=3-phenyl-, f=3-(2-furyl)-, t=3-(2-thienyl)-] with methanol or ethanol gave the corresponding methyl (Hxspme) or ethyl (Hxspee) esters. The reaction of these esters (HL) with triphenyltin(IV) hydroxide gave compounds of the type [SnPh3L], which were isolated and characterized as solids by elemental analysis, IR spectroscopy and mass spectrometry and in solution by multinuclear (1H, 13C and 119Sn) NMR spectroscopy. The structures of [SnPh3(pspme)], [SnPh3(fspme)] and [SnPh3(fspee)] were determined by X-ray diffractometry and the antimicrobial activity against E. coli, S. aureus, B. subtilis, P. aeruginosa, Resistant P. aeruginosa (a strain resistant to ‘carbapenem’), and C. albicans was tested and the in vitro cytotoxic activity against the HeLa-229, A2780 and A2780cis cell lines was determined for all compounds.

Inhibition of coiled coil domain containing protein 69 enhances platinum-induced apoptosis in ovarian cancer cells.

Cisplatin is a platinum-based drug that is used for the treatment of human gynecological cancers. However, molecular mechanisms of chemo-resistance in ovarian cancer are poorly understood. The aim of the study is to examine the role of coiled coil domain containing protein 69 (CCDC69) in the underlying mechanism of chemoresistance. Heavy CpG methylation (73.1% and 74.3%) was found in A2780 and A2780cis cells assessing by bisulfite sequencing. Restoration in the expression of CCDC69 was found in A2780 and A2780cis cells after 5-Aza-dC treatment. In fact, the expression levels of CCDC69 were about 3-4 fold higher in cisplatin-resistant A2780cis cells than its parental cisplatin-sensitive A2780 cells. When knockout CCDC69 in cisplatin-resistant A2780cis and SKOV3 cells by CRISPR/Cas9, the CCDC69 knockout cisplatin-resistant A2780cis and CCDC69 knockout SKOV3 cells were also shown increased sensitive to cisplatin treatment. Moreover, treating CCDC69 knockout A2780cis cells with cisplatin, abrogated G1 and G2/M arrest, increased of cleaved caspase 3&8, greater ΔΨm loss and higher levels of Bax were observed. When restoring CCDC69 expression in CCDC69 knockout A2780cis cells by transient transfection, it attenuated sensitivity to cisplatin. By immunoblotting, we found that depletion of CCDC69 increased p53 acetylation at K382 site and Bax mitochondrial redistribution. Additionally, inhibition of c-Myc enhanced cisplatin sensitivities in CCDC69 knockout A2780cis cells, overexpression of c-Myc reduced apoptosis in CCDC69 knockout SKOV3 cells. Our results showed that CCDC69 inhibition might interfere with the effectiveness of combination therapy with platinum drugs.

Abstract 3172: Inhibition of coiled coil domain containing protein 69 (CCDC69) enhance platinum-mediated apoptosis in ovarian cancer cells

Abstract Background and Objective:Molecular mechanisms of chemo-resistance in ovarian cancer are poorly understood. To identify gene involved in ovarian cancer chemo-resistance, 135 ovarian cancer patients with intact chemo-response information from The Cancer Genome Atlas (TCGA) database were included. Our analysis revealed that the level of CCDC69 mRNA is differentially expressed between chemo-sensitive group and chemo-resistant group. Moreover, there was a significant negatively correlation between CCDC69 promoter methylation and mRNA expression. The aim of the study is to examine the role of CCDC69 in the underlying mechanism of chemo-resistance. Methods: The expression levels of CCDC69 were detected in chemo-sensitive ovarian cancer A2780, chemo-resistant A2780cis and SKOV3 cell lines using quantitative RT-PCR and immunoblots. Promoter methylation status of CCDC69 were investigated by bisulfite sequencing. Silencing CCDC69 in A2780cis and SKOV3 cells were performed by Small-interfering RNAs (siRNAs) and CRISPR-Cas9. Cell viabilities after cisplatin treatment were evaluated by MTT and colony formation assays. Apoptosis was assessed by Annexin V/PI staining and caspases 3/8 activity. Cell cycle distributions and mitochondrial membrane potential (ΔΨm) were measured by flow cytometry. Relevant pathway proteins were determined by immunoblotting assays. Results: Heavy CpG methylation (73.1% and 74.3%) was found in A2780 and A2780cis cells. Restoration in the expression of CCDC69 were found in A2780 and A2780cis cells after 5-Aza-dC treatment. In fact, the expression levels of CCDC69 were about 3-4 fold higher in chemo-resistant A2780cis cells than its parental chemo-sensitive A2780 cells. Inhibition of CCDC69 in chemo-resistant A2780cis cells by si-RNA significantly increased sensitive to cisplatin treatment (p<0.05). When knockout CCDC69 in chemo-resistant A2780cis and SKOV3 cells by CRISPR-Cas9, the CCDC69KO chemo-resistant A2780cis and CCDC69KO SKOV3 cells were also shown increased sensitive to cisplatin treatment (p<0.001). Moreover, treating CCDC69KO A2780cis cells with cisplatin, abolished G1 and G2/M arrest, more cleaved caspase 3&8 activities, greater ΔΨm loss and higher levels of Bax were observed. When restoring CCDC69 expression in CCDC69KO A2780cis cells by transient transfection, it attenuated sensitivity to cisplatin. By immunoblotting, disruption of p14ARF-Mdm2-p53 cell cycle and/or apoptosis pathway, enhancement of MDM2 expression, and increase of p53 nuclear export were found in CCDC69KO A2780cis cells. Additionally, inhibition of c-Myc enhance cisplatin sensitivities in CCDC69KO A2780cis cells, overexpression of c-Myc reduce apoptosis in CCDC69KO SKOV3 cells. Summary: Our results show that silenced CCDC69 expression induced platinum-mediated apoptosis in ovarian cancer cells by abrogating c-Myc mediated apoptosis and cell cycle control networks. Citation Format: Long Cui, Liang Bo, Yihua Yang, Minhui Zhu, Joseph Kwong, Hongliang Zheng, Chi Chiu Wang. Inhibition of coiled coil domain containing protein 69 (CCDC69) enhance platinum-mediated apoptosis in ovarian cancer cells [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 3172. doi:10.1158/1538-7445.AM2017-3172

Abstract 1211: Lurbinectedin reverses platinum dependent IRF1 overexpression and nuclear localization, partially responsible for resistance to platinum drugs in ovarian cancer

Abstract Lurbinectedin (PM1183) is a new synthetic compound from the tetrahydroisoquinoline family, which has demonstrated a strong antiproliferative activity against a panel of human tumor models in preclinical assays and is currently being evaluated in phase III clinical trials in platinum-resistant ovarian cancer and small cell lung cancer. Lurbinectedin binds to DNA, inhibits trans-activated transcription, induces the degradation of elongating RNA Pol II and fools nucleotide excision repair to produce dsDNA breaks that need to be repaired mainly by homologous recombination (HR)1,2. Nearly 70% of patients diagnosed with ovarian cancer are in advanced stage, and the vast majority of them will eventually relapse after a primary cytoreductive surgery and several cycles of standard adjuvant chemotherapy including a platinum drug and a taxane. After a period of treatment with platinum drugs, patients will finally develop resistance, usually mediated by mechanisms such as drug detoxification or efflux and enhanced DNA repair. IRF-1 transcription factor expression has been shown to be up-regulated by cisplatin (CDDP) in ovarian cancer cells and might be limiting the response to the drug, likely by inhibiting cell proliferation3. Here we took advantage of the A2780/A2780cis human ovarian cancer cell lines, the second being a cisplatin resistant derivative, to investigate the role of IRF1 in the response of human ovarian cancer cells to cisplatin and lurbinectedin. A2780cis cells are, indeed, more resistant to cisplatin that their parental cell line but they do not differ in their resistance to lurbinectedin. Basal IRF-1 protein levels were actually higher in A2780cis cells than in their parental cell line, contributing to their resistance to cisplatin. Furthermore, cisplatin treatment induced the overexpression and nuclear localization of IRF-1 both, in A2780 and A2780cis cell lines. Contrarily, lurbinectedin did not induce the overexpression of IRF-1 neither in A2780 nor in A2780cis, explaining why this latter cell line is not resistant to the compound. Furthermore, lurbinectedin co-treatment with cisplatin reduced the expression of IRF-1 in A2780 and, more importantly, in A2780cis cells, explaining the synergism the combination has on these tumor cell lines. Thus, lurbinectedin not only did not activate the same mechanisms of resistance as cisplatin in ovarian cancer cells, but even reversed the resistance of these resistant cells to platinum drugs. 1 Santamaría Nuñez et al, 2016. Mol Cancer Ther 15(10):2399-2412 2 Romano et al, 2013. Int J Cancer. 2013 Nov;133(9):2024-33 3 Pavan et al, 2013. Eur J Cancer 49(4):964-973 Citation Format: Gema Santamaria Nuñez, Maria Jose Guillén, Juan F. Martínez-Leal, Pablo Avilés, Carlos M. Galmarini. Lurbinectedin reverses platinum dependent IRF1 overexpression and nuclear localization, partially responsible for resistance to platinum drugs in ovarian cancer [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 1211. doi:10.1158/1538-7445.AM2017-1211

Heparin antagonizes cisplatin resistance of A2780 ovarian cancer cells by affecting the Wnt signaling pathway.

Low molecular weight heparin (LMWH), the guideline based drug for prophylaxis and treatment of cancer-associated thrombosis, was recently shown to sensitize cisplatin resistant A2780cis human ovarian cancer cells for cisplatin cytotoxicity upon 24 h pretreatment with 50 μg × mL−1 of the LMWH tinzaparin in vitro, equivalent to a therapeutic dosage. Thereby, LMWH induced sensitization by transcriptional reprogramming of A2780cis cells via not yet elucidated mechanisms that depend on cellular proteoglycans. Here we aim to illuminate the underlying molecular mechanisms of LMWH in sensitizing A2780cis cells for cisplatin. Using TCF/LEF luciferase promotor assay (Top/Flash) we show that resistant A2780cis cells possess a threefold higher Wnt signaling activity compared to A2780 cells. Furthermore, Wnt pathway blockade by FH535 leads to higher cisplatin sensitivity of A2780cis cells. Glypican-3 (GPC3) is upregulated in A2780cis cells in response to LMWH treatment, probably as counter-regulation to sustain the high Wnt activity against LMWH. Hence, LMWH reduces the cisplatin-induced rise in Wnt activity and TCF-4 expression in A2780cis cells, but keeps sensitive A2780 cells unaffected. Consequently, Wnt signaling pathway appears as primary target of LMWH in sensitizing A2780cis cells for cisplatin toxicity. Considering the outstanding role of LMWH in clinical oncology, this finding appears as promising therapeutic option to hamper chemoresistance.

The Impact of the Low Molecular Weight Heparin Tinzaparin on the Sensitization of Cisplatin-Resistant Ovarian Cancers-Preclinical In Vivo Evaluation in Xenograft Tumor Models.

Resistance formation of tumors against chemotherapeutics is the major obstacle in clinical cancer therapy. Although low molecular weight heparin (LMWH) is an important component in oncology referring to guideline-based antithrombotic prophylaxis of tumor patients, a potential interference of LMWH with chemoresistance is unknown. We have recently shown that LMWH reverses the cisplatin resistance of A2780cis human ovarian cancer cells in vitro. Here we address the question whether this LMWH effect is also valid under in vivo conditions. Therefore, we established tumor xenografts of A2780 and cisplatin resistant A2780cis cells in nude mice and investigated the impact of daily tinzaparin applications (10 mg/kg BW) on anti-tumor activity of cisplatin (6 mg/kg BW, weekly) considering the tumor growth kinetics. Intratumoral platinum accumulation was detected by GF-AAS. Xenografts of A2780 and A2780cis cells strongly differed in cisplatin sensitivity. As an overall consideration, tinzaparin co-treatment affected the response to cisplatin of A2780cis, but not A2780 tumors in the later experimental time range. A subgroup analysis confirmed that initially smaller A2780cis tumors benefit from tinzaparin, but also small A2780 xenografts. Tinzaparin did not affect cisplatin accumulation in A2780cis xenografts, but strongly increased the platinum content in A2780, obviously related to morphological differences in both xenografts. Although we cannot directly confirm a return of A2780cis cisplatin resistance by tinzaparin, as shown in vitro, the present findings give reason to discuss heparin effects on cytostatic drug efficiency for small tumors and warrants further investigation.

Bis-picolinamide Ruthenium(III) Dihalide Complexes: Dichloride-to-Diiodide Exchange Generates Single trans Isomers with High Potency and Cancer Cell Selectivity.

A library of new bis-picolinamide ruthenium(III) dihalide complexes of the type [RuX2 L2 ] (X=Cl or I, L=picolinamide) have been synthesised and characterised. The complexes exhibit different picolinamide ligand binding modes, whereby one ligand is bound (N,N) and the other bound (N,O). Structural studies revealed a mixture of cis and trans isomers for the [RuCl2 L2 ] complexes but upon a halide exchange reaction to yield [RuI2 L2 ], only single trans isomers were detected. High cytotoxic activity against human cancer cell lines was observed, with the potencies of some complexes similar to or better than cisplatin. The conversion to [RuI2 L2 ] substantially increased the activity towards cancer cell lines by more than twelvefold. The [RuI2 L2 ] complexes displayed potent activity against the A2780cis (cisplatin-resistant human ovarian cancer) cell line, with a more than fourfold higher potency than cisplatin. Equitoxic activity was observed against normoxic and hypoxic cancer cells, which indicates the potential to eradicate both the hypoxic and aerobic fractions of solid tumours with similar efficiency. The activity of selected complexes against non-cancer ARPE-19 cells was also tested. The [RuI2 L2 ] complexes were found to be more potent than the [RuCl2 L2 ] analogues and also more selective towards cancer cells with a selectivity factor in excess of sevenfold.

Cisplatin resistance in cell models: evaluation of metallomic and biological predictive biomarkers to address early therapy failure.

Cisplatin, one of the most extensively used metallodrugs in cancer treatment, presents the important drawback of patient resistance. This resistance is the consequence of different processes including those preventing the formation of DNA adducts and/or their quick removal. Thus, a tool for the accurate detection and quantitation of cisplatin-induced adducts might be valuable for predicting patient resistance. To prove the validity of such an assumption, highly sensitive plasma mass spectrometry (ICP-MS) strategies were applied to determine DNA adduct levels and intracellular Pt concentrations. These two metal-relative parameters were combined with an evaluation of biological responses in terms of genomic stability (with the Comet assay) and cell cycle progression (by flow cytometry) in four human cell lines of different origins and cisplatin sensitivities (A549, GM04312, A2780 and A2780cis), treated with low cisplatin doses (5, 10 and 20 μM for 3 hours). Cell viability and apoptosis were determined as resistance indicators. Univariate linear regression analyses indicated that quantitation of cisplatin-induced G-G intra-strand adducts, measured 1 h after treatment, was the best predictor for viability and apoptosis in all of the cell lines. Multivariate linear regression analyses revealed that the prediction improved when the intracellular Pt content or the Comet data were included in the analysis, for all sensitive cell lines and for the A2780 and A2780cis cell lines, respectively. Thus, a reliable cisplatin resistance predictive model, which combines the quantitation of adducts by HPLC-ICP-MS, and their repair, with the intracellular Pt content and induced genomic instability, might be essential to identify early therapy failure.