Human Ovarian Carcinoma Cell Line Research Articles

Danusertib Induces Apoptosis, Cell Cycle Arrest, and Autophagy but Inhibits Epithelial to Mesenchymal Transition Involving PI3K/Akt/mTOR Signaling Pathway in Human Ovarian Cancer Cells.

Ovarian carcinoma (OC) is one of the most common gynecological malignancies, with a poor prognosis for patients at advanced stage. Danusertib (Danu) is a pan-inhibitor of the Aurora kinases with unclear anticancer effect and underlying mechanisms in OC treatment. This study aimed to examine the cancer cell killing effect and explore the possible mechanisms with a focus on proliferation, cell cycle progression, apoptosis, autophagy, and epithelial to mesenchymal transition (EMT) in human OC cell lines C13 and A2780cp. The results showed that Danu remarkably inhibited cell proliferation, induced apoptosis and autophagy, and suppressed EMT in both cell lines. Danu arrested cells in G2/M phase and led to an accumulation of polyploidy through the regulation of the expression key cell cycle modulators. Danu induced mitochondria-dependent apoptosis and autophagy in dose and time-dependent manners. Danu suppressed PI3K/Akt/mTOR signaling pathway, evident from the marked reduction in the phosphorylation of PI3K/Akt/mTOR, contributing to the autophagy inducing effect of Danu in both cell lines. In addition, Danu inhibited EMT. In aggregate, Danu exerts potent inducing effect on cell cycle arrest, apoptosis, and autophagy, but exhibits a marked inhibitory effect on EMT. PI3K/Akt/mTOR signaling pathway contributes, partially, to the cancer cell killing effect of Danu in C13 and A2780cp cells.

The expression and function of toll-like receptors (TLRs) in ovarian cancer cell lines

Meeting abstracts This study is to investigate which Toll Like Receptors (TLRs) are expressed on human ovarian cancer cell lines and to evaluate the effect of TLR expression on the growth of ovarian cancer cells. Four human ovarian epithelial carcinoma cell lines SKOV3 (clear), OVCAR3 (serous

Modulation of Multidrug Resistance Protein 2 Efflux in the Cisplatin Resistance Human Ovarian Carcinoma Cells A2780/RCIS by Sesquiterpene Coumarins.

Recent in vitro studies showed that sesquiterpene coumarins (SCs) can be used as chemosensitizers. In this study, 14 SCs were isolated and purified from roots of four Ferula species and their structures were elucidated by spectroscopic methods. The purified SCs were evaluated for multidrug resistance (MDR) reversal properties in A2780/RCIS cells (cisplatin-resistant derivatives of the human ovarian carcinoma cell line A2780P). Among the tested compounds, mogoltacin, mogoltadone, farnesiferol A, farnesiferol B, farnesiferol C, lehmferin, conferdione, and samarcandin showed significant MDR reversing effects. The combination of nontoxic concentrations of SCs (20 μM) with cisplatin enhanced cisplatin cytotoxicity on A2780/RCIS cells significantly. Flow cytometric efflux assay confirmed that the intracellular accumulation of 5-carboxyfluorescein diacetate (5-CFDA) was significantly increased in A2780/RCIS cells when treated with SCs. Our findings revealed that conferdione and samarcandin possessed the highest inhibitory effects on multidrug resistance-associated protein 2 pump efflux, and therefore, these compounds could be considered as lead scaffolds for further structure modifications.

Sphingolipids modulate the epithelial-mesenchymal transition in cancer.

Sphingolipids modulate the epithelial-mesenchymal transition in cancer.

Assessment of estrogenic activity in PM₁₀ air samples with the ERE-CALUX bioassay: Method optimization and implementation at an urban location in Flanders (Belgium).

Endocrine disrupting chemicals represent a broad class of compounds, are widespread in the environment and can pose severe health effects. The objective of this study was to investigate the overall estrogen activating potential of PM10 air samples at an urban location with high traffic incidence in Flanders, using a human in vitro cell bioassay. PM10 samples (n = 36) were collected on glass fiber filters every six days between April 2013 and January 2014 using a high-volume sampler. Extraction was executed with a hexane/acetone mixture before analysis using a recombinant estrogen-responsive human ovarian carcinoma (BG1Luc4E2) cell line. In addition, several samples and procedural blanks were extracted with ultra-pure ethanol or acetonitrile to compare extraction efficiencies. Results were expressed as bioanalytical equivalents (BEQs) in femtogram 17β-estradiol equivalent (fg E2-Eq) per cubic meter of air. High fluctuations in estrogenic activity were observed during the entire sampling period, with mean and median BEQs of 50.7 and 35.9 fg E2-Eq m(-)(3), respectively. Estrogenic activity was measured in more than 70% of the samples and several sample extracts showed both high BEQs and high cytotoxicity, which could not be related to black carbon, PM10 or heavy metal concentrations. At this moment, it remains unclear which substances cause this toxicity, but comparison of results obtained with different extraction solvents indicated that acetone/hexane extracts contained more compounds that were cytotoxic and suppressive of responses than those extracted using ultra-pure ethanol. Although more research is needed, the use of a more polar extraction solvent seems to be advisable.

Effects of cytochalasin congeners, microtubule-directed agents, and doxorubicin alone or in combination against human ovarian carcinoma cell lines in vitro.

BackgroundAlthough the actin cytoskeleton is vital for carcinogenesis and subsequent pathology, no microfilament-directed agent has been approved for cancer chemotherapy. One of the most studied classes of microfilament-directed agents has been the cytochalasins, mycotoxins known to disrupt the formation of actin polymers. In the present study, we sought to determine the effects of cytochalasin congeners toward human drug sensitive and multidrug resistant cell lines.MethodsSKOV3 human ovarian carcinoma and several multidrug resistant derivatives were tested for sensitivity against a panel of nine cytochalasin congeners, as well as three clinically approved chemotherapeutic agents (doxorubicin, paclitaxel, and vinblastine). In addition, verapamil, a calcium ion channel blocker known to reverse P-glycoprotein (P-gp) mediated drug resistance, was used in combination with multiple cytochalasin congeners to determine whether drug sensitivity could be increased.ResultsWhile multidrug resistant SKVLB1 had increased drug tolerance (was more resistant) to most cytochalasin congeners in comparison to drug sensitive SKOV3, the level of resistance was 10 to 1000-fold less for the cytochalasins than for any of the clinically approved agents. While cytochalasins did not appear to alter the expression of ATP binding cassette (ABC) transporters, several cytochalasins appeared to inhibit the activity of ABC transporter-mediated efflux of rhodamine 123 (Rh123), suggesting that these congeners do have affinity for drug efflux pumps. Cytochalasins also appeared to significantly decrease the F/G-actin ratio in both drug sensitive and drug resistant cells, indicative of marked microfilament inhibition. The cytotoxicity of most cytochalasin congeners could be increased with the addition of verapamil, and the drug sensitivity of resistant SKVLB1 to the clinically approved antineoplastic agents could be increased with the addition of cytochalasins. As assessed by isobolographic analysis and Chou-Talalay statistics, cytochalasin B and 21,22-dihydrocytochalasin B (DiHCB) demonstrated notable synergy with doxorubicin and paclitaxel, warranting further investigation in a tumor-bearing mammalian model.ConclusionCytochalasins appear to inhibit the activity of P-gp and potentially other ABC transporters, and may have novel activity against multidrug resistant neoplastic cells that overexpress drug efflux proteins.

Higher generation cationic N,N-ruthenium(II)-ethylene-glycol-derived metallodendrimers: Synthesis, characterization and cytotoxicity

A new series of higher generation cationic N,N-chelating, ruthenium(II)-ethylene-glycol metallodendrimers from first-, second-, third- and fourth-generation pyridylimine-based poly(propylene) dendrimer scaffolds of the type, DAB-(NH2)n (where n = 4, 8, 16 or 32, DAB = diaminobutane) have been prepared. Four metallodendrimers were synthesized, by first reacting DAB-(NH2)n with 2-pyridinecarboxaldehyde; and subsequently reacting the Schiff-base dendritic ligands with the [(η6-C6H5OCH2CH2OH)RuCl2]2 dimer, via a bridge-splitting reaction. All compounds were fully characterized using numerous spectroscopic and analytical techniques. In vitro anticancer activities of the complexes were evaluated against the A2780 cisplatin-sensitive and A2780cisR cisplatin-resistant human ovarian carcinoma cell lines and against the non-tumorigenic HEK-293 human embryonic kidney cell line, with the largest dendrimer being the most cytotoxic and selective of all the compounds. A mononuclear model analog was prepared to investigate the size dependency of the systems towards the biological activity.

MicroRNA-133b targets glutathione S-transferase π expression to increase ovarian cancer cell sensitivity to chemotherapy drugs.

BackgroundAccumulating studies reveal that aberrant microRNA (miRNA) expression can affect the development of chemotherapy drug resistance by modulating the expression of relevant target proteins. The aim of this study was to investigate the role of miR-133b in the development of drug resistance in ovarian cancer cells.MethodsWe examined the levels of miR-133b expression in ovarian carcinoma tissues and the human ovarian carcinoma cell lines (A2780, A2780/DDP and A2780/Taxol, respectively). We determined the cell viability of these cell lines treated with cisplatin or paclitaxel in the presence or absence of miR-133b or anti-miR-133b transfection using the MTT assay. Reverse transcription polymerase chain reaction and Western blotting were used to assess the mRNA and protein expression levels of two drug-resistance-related genes: glutathione S-transferase (GST)-π and multidrug resistance protein 1 (MDR1). The dual-luciferase reporter assay was used to detect the promoter activity of GST-π in the presence and absence of miR-133b.ResultsThe expression of miR-133b was significantly lower in primary resistant ovarian carcinomas than in the chemotherapy-sensitive carcinomas (P<0.05), and the same results were found in primary resistant ovarian cell lines (A2780/Taxol and A2780/DDP) compared to the chemotherapy-sensitive cell line (A2780; P<0.05). Following miR-133b transfection, four cell lines showed increased sensitivity to paclitaxel and cisplatin, while anti-miR-133b transfection reduced cell sensitivity to paclitaxel and cisplatin. Dual-luciferase reporter assay showed that miR-133b interacted with the 3′-untranslated region of GST-π. Compared to controls, the mRNA and protein levels of MDR1 and GST-π were downregulated after miR-133b transfection and upregulated after anti-miR-133b transfection.ConclusionMicroRNA-133b may reduce ovarian cancer drug resistance by silencing the expression of the drug-resistance-related proteins, GST-π and MDR1. In future, the combination of miR-133b with chemotherapy agents may prevent the development of drug resistance in ovarian cancers.

Dickkopf-3 (DKK-3) obstructs VEGFR-2/Akt/mTOR signaling cascade by interacting of β2-microglobulin (β2M) in ovarian tumorigenesis

In this study, we investigated a possible mechanism of β2-microglobulin (β2M) function in cancer metastases in vitro, using a human ovarian carcinoma cell line. β2M, a modulator acts as a cell growth-promoting and cellular signaling factors, was identified as a dickkopf-3 (DKK-3) interacting protein. We also observed that DKK-3 suppresses endothelial cell angiogenesis of β2M through vascular endothelial growth factor receptor-2 (VEGFR-2) in tumorigenesis. Luciferase activity was remarkably reduced by the transfection of DKK-3 in a dose-dependent manner. In addition, over-expression of β2M activates cell growth by suppressing DKK-3-induced apoptosis. The effect of β2M on cell cycle and apoptosis-regulatory components was also confirmed through the silencing of β2M expression. Furthermore, induction of β2M-mediated VEGFR-2/Akt/mTOR phosphorylation and tumor angiogenesis was significantly suppressed by over-expression of DKK-3. Taken together, our results suggest an underlying mechanism for an increase of β2M-related activity in ovarian tumor cells.

Abstract 4442: TP53 hot spot mutations in ovarian cancer: Selective resistance to microtubule stabilizing agents in monoclonal cells and comparison of clinical outcomes from The Cancer Genome Atlas data

Abstract TP53 is mutated in 96% of high grade serous epithelial ovarian carcinomas (HGS EOC) sequenced by The Cancer Genome Atlas (TCGA). Missense mutations at hot spot codons R273, R248, and R175 are the most common oncogenic TP53 mutations among ovarian cancers. To evaluate if TP53 hot spot mutations confer selective resistance to microtubule stabilizing agents (MSAs), the effects of MSAs on hot spot TP53 mutant ovarian carcinoma cell lines were determined. HGS EOCs that expressed one of the three most common TP53 hot spot missense mutations were also identified and studied using TCGA datasets. The human TP53 wild-type ovarian carcinoma cell line A2780 was stably transfected with an empty vector pCMV-neo or a missense TP53 mutation at hot spot codon 175 (m175), 248 (m248), or 273 (m273). Doubling times and inhibitory concentrations of each cell line treated with the MSAs paclitaxel, epoB, and ixabepilone were determined. Effects of epoB on TP53 expression, phosphorylation, and acetylation, as well as TP53-regulated expression of p21 and mdm2, were examined by Western blot analysis. Expression of TP53 target genes p21, GADD45, BAX, PIDD, NF-kB2, PAI-1, and MDR1 was also measured by RT-PCR. Available TCGA genomic and clinical datasets of HGS EOCs were used to identify patients whose tumors expressed one of the three most common TP53 hot spot mutations. Progression free and overall survival outcomes, TP53 expression levels, and available disease and treatment parameters of patients with the hot spot mutations were compared. PARP cleavage, cell growth and cytotoxicity studies demonstrate that the TP53 mutant cell line m248 is resistant to epoB and is not acetylated during epoB treatment. p21 and PAI-1 were induced by epoB in A2780 cells. Expression of p21, GADD45 and PAI-1 in mutant cell lines was generally down regulated. The cell line m273 is 2-3-fold resistant to paclitaxel, possibly due to high expression of MDR1. Patients identified from available TCGA ovarian cancer datasets whose tumors expressed a TP53 missense mutation at codon R273 (n = 15), R248 (n = 11), or R175 (n = 15) demonstrated 10.8, 12.3 and 17.8 months (p = 0.54) median progression free survival and 84.1, 36.1, and 60.6 months (p = 0.23) median overall survival outcomes, respectively. Differences in TP53 expression level, debulking status, disease stage and grade, and age at presentation between cases grouped according to hot spot mutation were not significant. Monoclonal human ovarian cancer cells with different TP53 hot spot mutations were selectively resistant to MSAs paclitaxel and epoB. Patients with HGS EOC and a TP53 hot spot mutation had comparable charactertistics and outcomes. Mutational studies in monoclonal cell lines may offer limited insight regarding clinical disease in HGS EOC considering the established high genetic instability and heterogeneity of HGS EOC. Citation Format: Brandon-Luke L. Seagle, Gerda Hofstteter, Chia-Ping Yang, Kevin Eng, Oluwatosin Odunsi-Akanji, Kunle Odunsi, Shohreh Shahabi. TP53 hot spot mutations in ovarian cancer: Selective resistance to microtubule stabilizing agents in monoclonal cells and comparison of clinical outcomes from The Cancer Genome Atlas data. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4442. doi:10.1158/1538-7445.AM2015-4442

Abstract 1642: Antitumor activity of lurbinectedin toward ovarian clear cell carcinoma

Abstract Objectives:Lurbinectedin is a marine-derived covalent DNA minor groove binder and is structurally similar to trabectedin except for the C subunit. Lurbinectedin (PM01183) is a newly developed antitumor agent that is currently being investigated in phase I/II clinical trials in patients with solid tumors including ovarian cancer. The objective of this study is to investigate the effectiveness of lurbinectedin as a single agent or as combination with existing antitumor agent against ovarian clear cell carcinoma which is regarded as an aggressive chemoresistant histological subtype. Methods:Using human ovarian clear cell carcinoma cell lines, the anti-tumor effects of lurbinectedin, SN38, topotecan, doxorubicin, cisplatin and paclitaxel as single agent were assessed by MTS assay. Then the cytotoxicities of combination treatments involving lurbinectedin and 1 of the 4 agents were evaluated by isobologram analysis to examine which of these combinations displayed synergistic effects. The antitumor activities of the combination treatments were also examined using cisplatin-resistant and paclitaxel-resistant ovarian clear cell carcinoma sublines, which were derived from the parental clear cell carcinoma cells by continuously exposing them to cisplatin or paclitaxel. Finally, using nude mice inoculated with clear cell carcinoma cells, we examined the growth-inhibitory effect of lurbinectedin in vivo. Results:Lurbinectedin showed significant antitumor activity toward chemosensitive and chemoresistant CCC cells in vitro. Mouse xenografts of CCC cells revealed that lurbinectedin significantly inhibits tumor growth. Concurrent exposure to lurbinectedin and SN-38 resulted in synergistic interactions in CCC cell lines. The combination of lurbinectedin plus SN-38 also had strong synergistic effects on both the cisplatin-resistant and paclitaxel-resistant CCC cell lines. Treatment with everolimus significantly enhanced the antitumor activity of single agent lurbinectedin and combination of lurbinectedin plus SN-38. Conclusions:Lurbinectedin is a promising agent for CCC as a first-line chemotherapy and as a second-line treatment of recurrent CCC that had previously been treated with cisplatin or paclitaxel. Moreover, lurbinectedin combined with irinotecan may be more efficacious for the management of CCC. Citation Format: Ryoko Takahashi, Seiji Mabuchi, Yuri Matsumoto, Mahiru Kawano, Tomoyuki Sasano, Hiromasa Kuroda, Katsumi Kozasa, Kenjirou Sawada, Tadashi Kimura. Antitumor activity of lurbinectedin toward ovarian clear cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1642. doi:10.1158/1538-7445.AM2015-1642

Abstract LB-274: Transcriptional factor Snail and MMP-9 signaling axis amplifies Neu1-MMP-9 crosstalk on EGF receptor to regulate tumor neovascularization, growth and metastasis in mouse model of human ovarian carcinoma

Abstract Snail is a transcriptional factor and repressor of E-cadherin. It is well known for its role in cellular invasion, epithelial to mesenchymal transition (EMT), tumor progression and metastases. shRNA lentiviral knockdown (KD) of Snail and its associated member Slug in human A2780 ovarian epithelial carcinoma cell line was investigated to identify its role in tumor neovascularization. Live cell sialidase, WST-1 and immunohistochemistry assays were used to evaluate sialidase activity, cell proliferation and the expression levels of tumor E-, N- and VE-cadherins, and host endothelial CD31+(PECAM-1) cells in A2780, A2780 Snail KD and A2780 Slug KD tumors grown in RAGxCγ double mutant mice. Oseltamivir phosphate (OP), anti-Neu1and MMP-9 specific inhibitor blocked Neu1 activity associated with epidermal growth factor (EGF) stimulated A2780 cells. Silencing Snail and Slug in A2780 cells abrogated the Neu1 activity following EGF stimulation of the cells compared to parental cells. OP treatment of A2780 and cisplatin-resistant A2780cis cells reproducibly and dose-dependently abated the cell viability with a LD50 of 7 and 4μM, respectively. Heterotopic xenografts of A2780 tumors developed abnormal bloody tumor vasculature in these mice. Anti-tumor activity of OP treatment at 50 mg/kg daily I.P. did not significantly impede A2780 tumor growth rate in a time-to-progression, but significantly cause a reduction of lung metastases compared with the untreated and OP 30 mg/kg cohorts. Silencing Snail in A2780 cells, but not the Slug KD member completely abrogated the tumor vascularization, growth and spread to the lungs in these mice. A2780 and A2780 Slug KD tumors expressed high levels of human N- and VE-cadherins, and host CD31+ endothelial cells. These findings uncover a novel organizational signaling platform connecting the Snail-MMP-9 signaling axis in amplifying the Neu1-MMP-9 cross-talk in regulating EGF receptors, tumor neovascularization, growth and invasiveness. Citation Format: Myron R. Szewczuk, Samar Abdulkhalek, Olivia Geen, Lacey Brodhagen, Fiona Haxho, Farah Alghamdi, Stephanie Allison, Duncan Simmons, Leah O'Shea, Ronald Neufeld. Transcriptional factor Snail and MMP-9 signaling axis amplifies Neu1-MMP-9 crosstalk on EGF receptor to regulate tumor neovascularization, growth and metastasis in mouse model of human ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-274. doi:10.1158/1538-7445.AM2015-LB-274

Abstract 2669: Chaetoglobosin K, an Akt pathway inhibitor, prevents proliferation and migration of prostate carcinoma cells

Abstract Chaetoglobosin K (ChK) is an indolylcytochalasin isolated from the fungus Diplodia macrospora that has shown anti-tumorigenic effects in multiple human lung and ovarian carcinoma cell lines. We have previously reported that ChK decreases Akt activation and modulates key downstream Akt/mTOR pathway effectors in tumorigenic cells. The down-regulation of Akt activation by ChK is independent of class I PI3K enzyme inhibition or the upstream mediators PDK1, PTEN, and mTORC2. The purpose of these studies was to investigate the effect of ChK on the proliferation and migration of prostate carcinoma cells of various metastatic potential and PTEN status. The activation of Akt was monitored to assess the ability of ChK to inhibit Akt in prostate carcinoma cells. In these studies, MTT assays were used to measure the number of viable PC3, DU145, and LNCaP cells treated with ChK. Scratch wound and transwell migration assays were utilized to examine the effect of ChK on cell migration. To assess Akt activation, western blot analysis was performed to measure changes in Akt phosphorylation at the ser473 and thr308 sites after cells were treated with ChK or vehicle. ChK was found to inhibit the number of viable PC3, DU145, and LNCaP cells with IC50 values in the 3-5μM range. ChK inhibited migration of PC3 cells into the wound space at 1μM by approximately 60% after 72 hours as compared to vehicle treated controls. ChK inhibited transwell migration of PC3 cells by greater than 90% compared to controls after 5 hours. Additionally, ChK treatment lowered Akt phosphorylation at key activation sites. In summary, ChK inhibits the proliferation, migration, and Akt activation of prostate carcinoma cells in vitro. Further mechanistic studies of ChK will substantiate its therapeutic potential in the treatment of prostate and other cancers. Citation Format: Amna Ali, Silvia Caggia, Diane F. Matesic, Shafiq A. Khan. Chaetoglobosin K, an Akt pathway inhibitor, prevents proliferation and migration of prostate carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2669. doi:10.1158/1538-7445.AM2015-2669

Tanshinone IIA Facilitates TRAIL Sensitization by Up-regulating DR5 through the ROS-JNK-CHOP Signaling Axis in Human Ovarian Carcinoma Cell Lines.

Tanshinone IIA (TIIA) extracted from Salvia miltiorrhiza has been shown to possess antitumor and TRAIL-sensitizing activity. The involvement of DR5 in the mechanism whereby TIIA exerts its effects is unknown. This study aimed to explore the mechanism underlying TIIA augmentation of TRAIL-induced cell death in ovarian carcinoma cells. Cell viability was determined by MTS assay. Real-time RT-PCR and Western blotting were used to assess the mRNA and protein expression of relating signaling proteins. Transcriptional activation was explored by a dual-luciferase reporter assay. We found that TIIA sensitized human ovarian carcinoma cells to TRAIL-induced extrinsic apoptosis. Combined treatment with subtoxic concentrations of TIIA and TRAIL was more effective than single treatments with respect to cytotoxicity, clonogenic inhibition, and the induction of caspase-8 and PARP activity in ovarian carcinoma cell lines TOV-21G and SKOV3. TIIA induced DR5 protein and mRNA expression in a concentration-dependent manner. DR5/Fc treatment markedly suppressed the TRAIL cytotoxicity enhanced by TIIA. These results indicate that DR5 plays an essential role in TIIA-induced TRAIL sensitization and that induction of DR5 by TIIA is mediated through the up-regulation of CCAAT/enhancer-binding protein homologous protein (CHOP). Knockdown of CHOP gene expression by shRNA attenuated DR5 up-regulation and rescued cell viability under the treatment of TIIA-TRAIL combination. TIIA promoted JNK-mediated signaling to up-regulated CHOP and thereby inducing DR5 expression as shown by the ability of a JNK inhibitor to potently suppress the TIIA-mediated activation of CHOP and DR5. In addition, the quenching of ROS using NAC prevented the induction of JNK phosphorylation and CHOP induction. Furthermore, inhibition of ROS by NAC significantly attenuated TRAIL sensitization by TIIA. Taken together, these data suggest that TIIA enhances TRAIL-induced apoptosis by upregulating DR5 receptors through the ROS-JNK-CHOP signaling axis in human ovarian carcinoma cells.

Cytotoxic effects of mithramycin DIG-MSK can depend on the rise of autophagy

DIG-MSK (demycarosil-3D-β-d-digitoxosyl mithramycin SK; EC-8042), a novel analogue of mithramycin A, induced autophagy in HCT116 human colon carcinoma and, to a lesser extent, in A2780 human ovarian carcinoma cell lines, which was followed by apoptosis and/or necrotic cell death in a time-dependent way. The effects of DIG-MSK included changes in the expression of a set of genes involved in autophagy, the progression of cells through the different phases of cell cycle, and their halting at the checkpoints. Cells treated with the glucose analogue 2-DG (2-deoxy-d-glucose), which induces autophagy because it impairs cell metabolism, or co-treated with 2-DG plus DIG-MSK, also showed altered gene expression and autophagy. In A2780 cells, some genes involved in autophagy were down-regulated by the different treatments, yet the levels of the proteins they encode could be enough to ensure autophagic flux. In HCT116 cells, up-regulation of several pro-autophagic genes resulted in strong autophagic response. Acidic cell organelles and autophagic flux were more evident in HCT116 than in A2780 cells. DIG-MSK was still cytotoxic in cells that underwent autophagy induced by 2-DG. Therefore, we verified that autophagy resulting from a stress response did not protect cells against DIG-MSK, but, instead, autophagy promoted by either 2-DG or the novel mithralogue can enhance the antitumour activity, which depended on the cell type.

Hypoxia-Sensitive Metal β-Ketoiminato Complexes Showing Induced Single-Strand DNA Breaks and Cancer Cell Death by Apoptosis.

A series of ruthenium and iridium complexes have been synthesized and characterized with 20 novel crystal structures discussed. The library of β-ketoiminato complexes has been shown to be active against MCF-7 (human breast carcinoma), HT-29 (human colon carcinoma), A2780 (human ovarian carcinoma), and A2780cis (cisplatin-resistant human ovarian carcinoma) cell lines, with selected complexes' being more than three times as active as cisplatin against the A2780cis cell line. Selected complexes were also tested against the noncancerous ARPE-19 (retinal pigment epithelial cells) cell line, in order to evaluate the complexes selectivity for cancer cells. Complexes have also been shown to be highly active under hypoxic conditions, with the activities of some complexes increasing with a decrease in O2 concentration. The enzyme thioredoxin reductase is overexpressed in cancer cells, and complexes reported herein have the advantage of inhibiting this enzyme, with IC50 values measured in the nanomolar range. The anticancer activity of these complexes was further investigated to determine whether activity is due to effects on cellular growth or cell survival. The complexes were found to induce significant levels of cancer cell death by apoptosis with levels induced correlating closely with activity in chemosensitivity studies. As a possible cause of cell death, the ability of the complexes to induce damage to cellular DNA was also assessed. The complexes failed to induce double-strand DNA breaks or DNA cross-linking but induced significant levels of single-strand DNA breaks, indicating a mechanism of action different from that of cisplatin.

Lentivirus-Mediated RNAi silencing targeting ERCC1 reverses cisplatin resistance in cisplatin-resistant ovarian carcinoma cell line.

The aim of the study was to investigate the potential mechanisms that interferencing of excision repair cross-complementing gene 1 (ERCC1) mediated by lentiviral vector in cisplatin-resistant ovarian cancer SKOV3/DDP cells. The human platinum-resistant ovarian carcinoma cell line SKOV3/DDP was transfected by pLVX-shRNA lentivirus. Interference efficiency for ERCC1 by lentiviruses transfection was detected by real-time polymerase chain reaction and western blot assay. CCK-8 assay was used for cell proliferation on cell resistance after transfection with ERCC1. Effects of cell apoptosis and cell cycles were detected by flow cytometry. The expression levels of ERCC1 were significantly suppressed in SKOV3/DDP cells after stably transfecting with shERCC1-recombinant plasmid. The results of cell viability assay demonstrated that interference with ERCC1 gene increased the sensitivity of SKOV3/DDP cells to cisplatin (p<0.01). ERCC1 gene-specific silencing promoted cell apoptosis of SKOV3/DDP cells (p<0.01) detected by flow cytometry. Cell cycle analysis results showed that the proportion of cells in G1 and S phase decreased, while the proportion of G2 phase cells increased in ERCC1-silencing cells. The differences were statistically significant (p<0.01), which demonstrated that stable interferencing with ERCC1 induced the cells arrest in G2 phase after being treated by DDP and silencing the expression of ERCC1 inhibited cell proliferation by preventing the progression of cell mitosis. ERCC1 gene silencing effectively reversed SKOV3/DDP cell resistance to cisplatin and increased sensitivity to cisplatin resistance in cisplatin-resistant ovarian cancer cells. Interference of ERCC1 promoted the apoptosis of SKOV3/DDP cells and prevented cell mitosis by inducing G0/G1 phase arrest. Thus, ERCC1 could be a potential therapeutic target for the therapy of cisplatin-resistant ovarian cancer and it would provide new ideas for epigenetic therapy of drug-resistant epithelial ovarian cancer.

Multiplexed tracking of combinatorial genomic mutations in engineered cell populations.

Multiplexed genome engineering approaches can be used to generate targeted genetic diversity in cell populations on laboratory timescales, but methods to track mutations and link them to phenotypes have been lacking. We present an approach for tracking combinatorial engineered libraries (TRACE) through the simultaneous mapping of millions of combinatorially engineered genomes at single-cell resolution. Distal genomic sites are assembled into individual DNA constructs that are compatible with next-generation sequencing strategies. We used TRACE to map growth selection dynamics for Escherichia coli combinatorial libraries created by recursive multiplex recombineering at a depth 10(4)-fold greater than before. TRACE was used to identify genotype-to-phenotype correlations and to map the evolutionary trajectory of two individual combinatorial mutants in E. coli. Combinatorial mutations in the human ES2 ovarian carcinoma cell line were also assessed with TRACE. TRACE completes the combinatorial engineering cycle and enables more sophisticated approaches to genome engineering in both bacteria and eukaryotic cells than are currently possible.

Synergistic effect of pH-responsive folate-functionalized poloxamer 407-TPGS-mixed micelles on targeted delivery of anticancer drugs.

BackgroundDoxorubicin (DOX), an anthracycline anticancer antibiotic, is used for treating various types of cancers. However, its use is associated with toxicity to normal cells and development of resistance due to overexpression of drug efflux pumps. Poloxamer 407 (P407) and vitamin E TPGS (D-α-tocopheryl polyethylene glycol succinate, TPGS) are widely used polymers as drug delivery carriers and excipients for enhancing the drug retention times and stability. TPGS reduces multidrug resistance, induces apoptosis, and shows selective anticancer activity against tumor cells. Keeping in view the problems, we designed a mixed micelle system encapsulating DOX comprising TPGS for its selective anticancer activity and P407 conjugated with folic acid (FA) for folate-mediated receptor targeting to cancer cells.MethodsFA-functionalized P407 was prepared by carbodiimide crosslinker chemistry. P407-TPGS/FA-P407-TPGS-mixed micelles were prepared by thin-film hydration method. Cytotoxicity of blank micelles, DOX, and DOX-loaded micelles was determined by alamarBlue® assay.ResultsThe size of micelles was less than 200 nm with encapsulation efficiency of 85% and 73% for P407-TPGS and FA-P407-TPGS micelles, respectively. Intracellular trafficking study using nile red-loaded micelles indicated improved drug uptake and perinuclear drug localization. The micelles show minimal toxicity to normal human cell line WRL-68, enhanced cellular uptake of DOX, reduced drug efflux, increased DOX–DNA binding in SKOV3 and DOX-resistant SKOV3 human ovarian carcinoma cell lines, and enhanced in vitro cytotoxicity as compared to free DOX.ConclusionFA-P407-TPGS-DOX micelles show potential as a targeted nano-drug delivery system for DOX due to their multiple synergistic factors of selective anticancer activity, inhibition of multidrug resistance, and folate-mediated selective uptake.

Isolation and cytotoxicity evaluation of taxanes from the barks of Taxus wallichiana var. mairei.

Fifteen taxanes (1-15) including a new taxane glucoside, 7β,9α,10β-triacetoxy-13α-hydroxy-5α-O-(β-d-glucopyranosyl)taxa-4(20),11-diene (1), were isolated from the barks of Taxus wallichiana var. mairei. Compounds 1-15 representing three sub-types of 6/8/6-taxane were evaluated in vitro for anti-proliferative activity against a panel of parental and drug-resistant cancer cells. Potent compounds were found while several exhibited selective cytotoxicity. Especially, 3, 8, and 10 showed selective inhibition to breast carcinoma cell line MCF-7, while 13 selectively inhibited taxol resistant human ovarian carcinoma cell line A2780/TAX (IC50=0.19μM), being more potent than the clinical drugs taxol (IC50=4.4μM) and docetaxol (IC50=0.42μM), and less cytotoxic to mouse embryonic fibroblast cell line NIH-3T3, a cell line close to normal cell line. The possible P-glycoprotein evasion mechanism of 13 against A2780/TAX and the preliminary structure-activity relationships (SARs) of this group of compounds were also discussed.