Cisplatin-resistant Ovarian Cancer Cells Research Articles

MiR-205-5p Mediated Downregulation of PTEN Contributes to Cisplatin Resistance in C13K Human Ovarian Cancer Cells.

Cisplatin resistance is a major cause of treatment failure in advanced ovarian cancer. The limited evidence shows the paradoxical regulation of miR-205 on chemotherapy resistance in cancer. Herein, we found that miR-205-5p was enormously increased in cisplatin-resistant C13K ovarian cancer cells compared with its cisplatin-sensitive OV2008 parental cells using miRNA microarrays, which was further verified by quantitative PCR. Furthermore, we confirmed that inhibition of miR-205-5p upregulated PTEN and subsequently attenuated its downstream target p-AKT, which inversed C13K cells from cisplatin resistance to sensitivity. Our data suggest that miR-205-5p contributes to cisplatin resistance in C13K ovarian cancer cells may via targeting PTEN/AKT pathway.

Luteolin sensitizes the antitumor effect of cisplatin in drug-resistant ovarian cancer via induction of apoptosis and inhibition of cell migration and invasion

Luteolin, a polyphenolic flavone, has been demonstrated to exert anti-tumor activity in various cancer types. Cisplatin drug resistance is a major obstacle in the management of ovarian cancer. In the present study, we investigated the chemo-sensitizing effect of luteolin in both cisplatin-resistant ovarian cancer cell line and a mice xenotransplant model. In vitro, CCK-8 assay showed that luteolin inhibited cell proliferation in a dose-dependent manner, and luteolin enhanced anti-proliferation effect of cisplatin on cisplatin-resistant ovarian cancer CAOV3/DDP cells. Flow cytometry revealed that luteolin enhanced cell apoptosis in combination with cisplatin. Western blotting and qRT-PCR assay revealed that luteolin increased cisplatin-induced downregulation of Bcl-2 expression. In addition, wound-healing assay and Matrigel invasion assay showed that luteolin and cisplatin synergistically inhibited migration and invasion of CAOV3/DDP cells. Moreover, in vivo, luteolin enhanced cisplatin-induced reduction of tumor growth as well as induction of apoptosis. We suggest that luteolin in combination with cisplatin could potentially be used as a new regimen for the treatment of ovarian cancer.

Seco-4-methyl-DCK derivatives as potent chemosensitizers

Twenty-five seco-4-methyl-DCK derivatives were designed, synthesized and evaluated for chemoreversal activity when combined with paclitaxel or vincristine in two drug-resistant cancer cell lines (A2780/T and KB-V) respectively. Most of the new compounds displayed moderate to significant MDR reversal activities in the P-gp overexpressing A2780/T and KB-V cells. Especially, compounds 7o and 7y showed the most potent chemosensitization activities with more than 496 and 735 reversal ratios at a concentration of 10 μM. Unexpectedly the newly synthesized compounds did not show chemosensitization activities observed in a non-P-gp overexpressing cisplatin resistant human ovarian cancer cell line (A2780/CDDP), implying that the MDR reversal effects might be associated with P-gp overexpression. Moreover, these compounds did not exhibit significant antiproliferative activities against nontumorigenic cell lines (HUVEC, HOSEC and T29) compared to the positive control verapamil at the tested concentration, which suggested better safety than verapamil. The pharmacological actions of the compounds will be studied further to explore their merit for development as novel candidates to overcome P-gp mediated MDR cancer.

Carbon monoxide sensitizes cisplatin-resistant ovarian cancer cell lines toward cisplatin via attenuation of levels of glutathione and nuclear metallothionein

Cisplatin resistance remains a major impediment to effective treatment of ovarian cancer. Despite initial platinum responsiveness, thiol-containing peptides and proteins, glutathione (GSH) and metallothionein (MT), bind and inactivate cisplatin in cancer cells. Indeed, high levels of GSH and MT in ovarian cancers impart cisplatin resistance and are predictive of poor prognosis. Cystathionine β-synthase (CBS), an enzyme involved in sulfur metabolism, is overexpressed in ovarian cancer tissues and is itself associated with cisplatin resistance. Treatment with exogenous carbon monoxide (CO), a known inhibitor of CBS, may mitigate cisplatin resistance in ovarian cancer cells by attenuation of GSH and MT levels. Using a photo-activated CO-releasing molecule (photoCORM), [Mn(CO)3(phen)(PTA)]CF3SO3 (phen = 1,10-phenanthroline, PTA = 1,3,5-triza-7-phosphaadamantane) we assessed the ability of CO to sensitize established cisplatin-resistant ovarian cancer cell lines to cisplatin. Cisplatin-resistant cells, treated with both cisplatin and CO, exhibited significantly lower cell viability and increased poly (ADP-ribose) polymerase (PARP) cleavage versus those treated with cisplatin alone. These cisplatin-resistant cell lines overexpressed CBS and had increased steady state levels of GSH and expression of nuclear MT. Both CO treatment and lentiviral-mediated silencing of CBS attenuated GSH and nuclear MT expression in cisplatin resistant cells. We have demonstrated that CO, delivered from a photoCORM, sensitizes established cisplatin-resistant cell lines to cisplatin. Furthermore, we have presented strong evidence that the effects of CO in circumventing chemotherapeutic drug resistance is at least in part mediated by the inactivation of endogenous CBS.

The cisplatin-induced lncRNA PANDAR dictates the chemoresistance of ovarian cancer via regulating SFRS2-mediated p53 phosphorylation

As a component of p53-dependent lncRNA (long non-coding RNA), PANDAR (the promoter of CDKN1A antisense DNA damage activated RNA) participates in the epigenetic regulation in human cancer. However, the involvement of PANDAR in cancer chemoresistance is unknown. In this study, we report that PANDAR serves as a negative regulator of cisplatin sensitivity in human ovarian cancer via PANDAR-SRFS2-p53 feedback regulation in nuclear. Our data showed that among the drugs commonly used in ovarian cancer therapy, cisplatin induces higher levels of PANDAR compared with doxorubicin and paclitaxel. We also proved that PANDAR exhibited higher expression in cisplatin-resistant ovarian cancer tissues and cells, compared with cisplatin-sensitive ones, and this expression pattern depends on wild-type p53 (wt-p53), not mutant-p53 (mt-p53). In vitro and in vivo, PANDAR overexpression improved cell survival rate and tumor growth in response to cisplatin, while depletion of PANDAR leads to a reduced tumor growth. Further investigation revealed that PANDAR-reduced cisplatin sensitivity was likely or partly due to the PANDAR-binding protein SFRS2 (arginine/serine-rich 2), a splicing factor with the ability to negative regulate p53 and its phosphorylation at Serine 15 (Ser15). This feedback regulation of PANDAR–SFRS2–p53 leads to a reduced transactivation of p53-related pro-apoptotic genes, such as PUMA (p53-upregulated modulator of apoptosis). In addition, in platinum-treated patients with relapsed ovarian cancer, resistant period was positively correlated with the expression of PANDAR and SFRS2, and inversely associated with expression of p53-Ser15 and PUMA in these clinical tissues. Last but not least, the role of PANDAR in chemoresistance was confirmed in patients with ovarian cancer. These findings reveal a novel regulatory maneuver of cancer cells in response to chemostress, and might shed light on overcoming cisplatin resistance in ovarian cancer.

Cell growth inhibitory effects of polyphenols with naphthalene skeleton against cisplatin-resistant ovarian cancer cells

Cisplatin often shows the drug resistance which could limit the chemotherapeutic efficacy. Thus, it is necessary to develop anticancer agents against cisplatin-resistant cancer cells. To identify pharmacophores exhibiting the cell growth inhibitory effect against cisplatin-resistant A2780/Cis ovarian cancer cells, we prepared 35 synthetic polyphenols bearing naphthalene skeleton including naphthalenyl chalcones, naphthalenyl flavones, naphthalenyl flavanones, 4,5-dihydro-1H-pyrazol-3-yl)naphthalen-2-ols, naphthalen-1-yl-N-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamides, and 4,5-dihydro-1H-pyrazol-3-yl)naphthalen-1-ol. The correlation between their inhibitory effects and structural properties was evaluated using hologram quantitative structure activity relationship and comparative molecular field analysis. The pharmacophores derived here can lead us to design new polyphenols against the growth of cisplatin-resistant cells.

Self-Assembled Honokiol-Loaded Microbubbles in the Treatment of Ovarian Cancer by Ultrasound Irradiation.

To observe the growth inhibition of subcutaneous ovarian cancer xenografts in nude mice and mechanisms by ultrasound irradiation with honokiol-loaded poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL, PCEC) microbubbles (HK-PCEC-MB) as a promising therapeutic approach to ovarian tumor. HK-PCEC-MB were prepared using the double-emulsion solvent evaporation technique. Particles were characterized with regard to shape, size, distribution and surface potential using dynamic light scattering and the Malvern Zetasizer ZS90 system. Entrapment efficiency and loading amounts of honokiol were determined via high-performance liquid chromatography (HPLC). Pharmacological activity and optimal mode of action were examined using MTT assay. Nude mice were sorted into five groups of cisplatin-sensitive (A2780s) and cisplatin-resistant (A2780cp) ovarian cancer cell subcutaneous xenograft models: (1) HK-PCEC-MB+ultrasound; (2) HK; (3) PCEC-MB+ultrasound; (4) HK-PCEC-MB; (5) controls. We observed rates of growth inhibition, necrosis, and apoptosis, as well as microvessel density (MVD) changes. Physical properties of HK-PCEC-MB followed the normal pattern. The drug entrapment efficiency of HK-PCEC-MB was 65.08±2.31% and drug loading amount was 6.51±0.23%. Cancer cell survival rate was lowest in vitro at a drug concentration of 10 μg/mL with exposure to 2.58 W ultrasonic wave. In ultrasound irradiation combined with HK-PCEC-MB group, rates of necrosis and apoptosis were the highest, while the number of microvessel was the least. We have successfully prepared self-assembled HK-PCEC-MB that inhibits resistant human ovarian tumor growth by ultrasound irradiation.

Morin decreases galectin-3 expression and sensitizes ovarian cancer cells to cisplatin

PurposeThis study aimed at evaluating whether morin (a natural flavonoid and a known inhibitor of NF-κB) can sensitize ovarian cancer cells to cisplatin by decreasing the expression of galectin-3, which is an anti-apoptotic protein regulated by NF-κB transcription factor.MethodsTo assess the possibility of augmentation the activity of cisplatin by morin, we studied the separate and the combined effect of morin and cisplatin on viability, proliferation, and apoptosis of TOV-21G (cisplatin-sensitive) and SK-OV-3 (cisplatin-resistant) ovarian cancer cells. We also analysed the effect of morin and cisplatin on galectin-3 expression at the mRNA and protein levels.ResultsWe demonstrated that morin possess antitumor activity against TOV-21G and SK-OV-3 ovarian cancer cells by reducing cell viability and proliferation as well as increasing the induction of apoptosis. Co-treatment of the cells with selected concentrations of morin and cisplatin, accordingly to specific treatment approaches, reveals a synergism, which leads to sensitization of the cells to cisplatin. During this sensitization, morin significantly reduces the expression of galectin-3 at the mRNA and protein level, regardless of the presence of cisplatin.ConclusionsMorin sensitizes TOV-21G and SK-OV-3 ovarian cancer cells to cisplatin, what is associated with a decrease of the expression of galectin-3.

Pyruvate dehydrogenase kinase 1 contributes to cisplatin resistance of ovarian cancer through EGFR activation.

Patients with ovarian cancer frequently develop acquired drug resistance after the long-term chemotherapy, leading to disease progression. Enhanced epithelial-mesenchymal transition (EMT) has been implicated in chemoresistance of ovarian cancer cells; however, the molecular mechanisms involved are largely undefined. Pyruvate dehydrogenase kinase 1 (PDK1), a key regulatory enzyme in glucose metabolism, has been recognized as a gatekeeper of the Warburg effect, a hallmark of cancer. In this study, the function of PDK1 in cisplatin resistance of ovarian cancer in terms of growth and EMT was investigated. PDK1 was upregulated in cisplatin-resistant ovarian cancer cells. PDK1 knockdown in resistant cells led to increased sensitivity to cisplatin-induced cell death and apoptosis. PDK1 downregulation also reversed the EMT and cell motility in cisplatin-resistant cells. In a mouse xenograft model, tumors derived from PDK1-silenced ovarian cancer cells exhibited decreased tumor growth and EMT compared with control after the cisplatin treatment. Mechanistically, PDK1 overexpression led to increased phosphorylation of EGFR, and blocking EGFR kinase activity by erlotinib reversed cisplatin resistance induced by PDK1 overexpression. Furthermore, in patients with ovarian cancer, higher PDK1 and p-EGFR levels were associated with chemoresistance. These results supported that PDK1 contributes to chemoresistance of ovarian cancer by activating EGFR. Therefore, PDK1 may serve as a promising target to combat chemoresistance of ovarian cancer.

Cell-Permeable Bicyclic Peptidyl Inhibitors against NEMO-IκB Kinase Interaction Directly from a Combinatorial Library.

Macrocyclic peptides are capable of binding to flat protein surfaces such as the interfaces of protein-protein interactions with antibody-like affinity and specificity, but generally lack cell permeability in order to access intracellular targets. In this work, we designed and synthesized a large combinatorial library of cell-permeable bicyclic peptides, in which the first ring consisted of randomized peptide sequences for potential binding to a target of interest, while the second ring featured a family of different cell-penetrating motifs, for both cell penetration and target binding. The library was screened against the IκB kinase α/β (IKKα/β)-binding domain of NF-κB essential modulator (NEMO), resulting in the discovery of several cell-permeable bicyclic peptides, which inhibited the NEMO-IKKβ interaction with low μM IC50 values. Further optimization of one of the hits led to a relatively potent and cell-permeable NEMO inhibitor (IC50 = 1.0 μM), which selectively inhibited canonical NF-κB signaling in mammalian cells and the proliferation of cisplatin-resistant ovarian cancer cells. The inhibitor provides a useful tool for investigating the biological functions of NEMO/NF-κB and a potential lead for further development of a novel class of anti-inflammatory and anticancer drugs.

MiR-137 mediates the functional link between c-Myc and EZH2 that regulates cisplatin resistance in ovarian cancer.

Platinum drugs are used in first-line to treat ovarian cancer, but most of the patients eventually generate resistance after treatment with these drugs. Although both c-Myc and EZH2 have been implicated in regulating cisplatin resistance in ovarian cancer, the interplay between these two regulators is poorly understood. Using RNA sequence analysis (RNA-seq), for the first time we find that miR-137 level is extremely low in cisplatin resistant ovarian cancer cells, correlating with higher levels of c-Myc and EZH2 expression. Further analyses indicate that in resistant cells c-Myc enhances the expression of EZH2 by directly suppressing miR-137 that targets EZH2 mRNA, and increased expression of EZH2 activates cellular survival pathways, resulting in the resistance to cisplatin. Inhibition of c-Myc-miR-137-EZH2 pathway re-sensitizes resistant cells to cisplatin. Both in vivo and in vitro analyses indicate that cisplatin treatment activates c-Myc-miR-137-EZH2 pathway. Importantly, elevated c-Myc-miR-137-EZH2 pathway in resistant cells is sustained by dual oxidase maturation factor 1 (DUOXA1)-mediated production of reactive oxygen species (ROS). Significantly, clinical studies further confirm the activated c-Myc-miR-137-EZH2 pathway in platinum drug-resistant or recurrent ovarian cancer patients. Thus, our studies elucidate a novel role of miR-137 in regulating c-Myc-EZH2 axis that is crucial to the regulation of cisplatin resistance in ovarian cancer.

PTGER3 Induces Ovary Tumorigenesis and Confers Resistance to Cisplatin Therapy Through Up-Regulation Ras-MAPK/Erk-ETS1-ELK1/CFTR1 Axis

Inflammatory mediator prostaglandin E2-prostaglandin E2 receptor EP3 (PTGER3) signaling is critical for tumor-associated angiogenesis, tumor growth, and chemoresistance. However, the mechanism underlying these effects in ovarian cancer is not known. An association between higher tumoral expression of PTGER3 and shorter patient survival in the ovarian cancer dataset of The Cancer Genome Atlas prompted investigation of the antitumor effects of PTGER3 downmodulation. PTGER3 mRNA and protein levels were higher in cisplatin-resistant ovarian cancer cells than in their cisplatin-sensitive counterparts. Silencing of PTGER3 via siRNA in cancer cells was associated with decreased cell growth and less invasiveness, as well as cell-cycle arrest and increased apoptosis, mediated through the Ras-MAPK/Erk-ETS1-ELK1/CFTR1 axis. Furthermore, sustained PTGER3 silencing with multistage vector and liposomal 2'-F-phosphorodithioate-siRNA-mediated silencing of PTGER3 combined with cisplatin resulted in robust antitumor effects in cisplatin-resistant ovarian cancer models. These findings identify PTGER3 as a potential therapeutic target in chemoresistant ovarian cancers expressing high levels of this oncogenic protein. Funding: This work was supported in part by grants from the National Institutes of Health/National Cancer Institute (R44GM086937, P50 CA093459, U54 CA151668, P50 CA083639, P50 CA098258, R21 CA180145, UH3TR000943, RO1CA160687 and U54 CA96300), the Cancer Prevention Research Institute of Texas (RP120214), the FRANK McGraw Memorial Chair in Cancer Research, and the American Cancer Society Research Professor Award. This research was performed in the Flow Cytometry & Cellular Imaging Facility, which is supported in part by the National Institutes of Health through M. D. Anderson's Cancer Center Support Grant CA016672. Declaration of Interest: The authors have no potential conflicts of interest to disclose. X. Yang is a full-time employee at AM Biotechnologies. Ethical Approval: Animal studies were conducted in accordance with the guidelines set forth by the American Association for Accreditation of Laboratory Animal Care and the US Public Health Service policy on Humane Care and Use of Laboratory Animals. All mouse studies were approved and supervised by The University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee.

Small-molecule compounds targeting the STAT3 DNA-binding domain suppress survival of cisplatin-resistant human ovarian cancer cells by inducing apoptosis

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) plays important roles in oncogenic occurrence and transformation by regulating the expression of diverse downstream target genes important for tumor growth, metastasis, angiogenesis and immune evasion. Feasibility of targeting the DNA-binding domain (DBD) of STAT3 has been proven previously. With the aid of 3D shape- and electrostatic-based drug design, we identified a new STAT3 inhibitor, LC28, and its five analogs, based on the pharmacophore of a known STAT3 DBD inhibitor. Microscale thermophoresis assay shows that these compounds inhibits STAT3 binding to DNA with a Ki value of 0.74–8.87 μM. Furthermore, LC28 and its analogs suppress survival of cisplatin-resistant ovarian cancer cells by inhibiting STAT3 signaling and inducing apoptosis. Therefore, these compounds may serve as candidate compounds for further modification and development as anticancer therapeutics targeting the DBD of human STAT3 for treatment of cisplatin-resistant ovarian cancer.

Nanosecond Electric Pulses Induce Early and Late Phases of DNA Damage and Cell Death in Cisplatin-Resistant Human Ovarian Cancer Cells.

Chemoresistance is a challenge for management of ovarian cancer, and therefore the response of resistant cells to nanosecond electric pulses (nsEP) was explored. Human ovarian cancer cell line COC1 and the cisplatin-resistant subline COC1/DDP were subjected to nsEP (32 ns, 10 kV/cm, 10 Hz pulse repletion frequency, and 10 min exposure duration), and then the cellular responses were followed. The percentages of dead cells and of comet-formed cells in the alkaline assay displayed two peak levels (i.e., 2 and 8 h after nsEP exposure), with the highest value noted at 8 h; the percentage of comet-formed cells in the neutral assay was increased at 8 h; the apoptotic percentage was increased at 8 h, with collapse of the mitochondrial membrane potential and the activation of caspase-3 and caspase-9. The comet assay demonstrated DNA single-strand break at 2 h and double-strand break at 8 h. nsEP resulted in lower cytotoxicity in COC1/DDP cells compared with COC1 cells. These findings indicated that nsEP induced early and late phases of DNA damage and cell death, and these two types of cell death may have distinct applications to treatments of chemoresistant ovarian cancers.

Abstract B67: Antitumor effect of black tea pigments, theaflavin-3/3’-gallate, against cisplatin-resistant ovarian cancer cells

Abstract Ovarian cancer ranks fifth in cancer deaths among women in the United States, accounting for approximately 5% of all cancer deaths. Acquired resistance to conventional platinum-based chemotherapy drives the development of more selective anticancer drugs. Theaflavin-3-gallate (TF2a) and theaflavin-3’-gallate (TF2b) are the major bioactive components in black tea. In this study, we evaluated TF2a and TF2b as potential anticancer agents using human ovarian cancer cells. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37˚C in a humidified incubator with 5% CO2. The [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, flow cytometry, and Western blot analysis were used to confirm that TF2a and TF2b exhibited a potent growth-inhibitory effect against cisplatin-resistant ovarian cancer A2780/CP70 cells. Treatment with TF2a and TF2b (10-40 μM) significantly decreased A2780/CP70 cell viability (p< 0.05). Treatment with TF2a and TF2b (5, 10 and 20 μM) increased the percentage of apoptotic cells in a concentration-dependent manner. Western blot analysis was used to evaluate the expression of proteins related to apoptosis in A2780/CP70 cells treated with designated concentrations (0, 5, 10, and 20 μM) of TF2a and TF2b for 24 h. The protein levels of procaspase-3/7 for TF2a-treated cells and procaspase 3 for TF2b-treated cells decreased significantly in A2780/CP70 cells (p< 0.05). Treatment with TF2a or TF2b significantly increased the protein levels of cleaved caspase-3/7, full-length PARP, and cleaved PARP in A2780/CP70 cells. Our study demonstrated that TF2a and TF2b are potential agents for treating platinum-resistant ovarian cancer by inducing apoptosis. Note: This abstract was not presented at the conference. Citation Format: Yi Charlie Chen. Antitumor effect of black tea pigments, theaflavin-3/3’-gallate, against cisplatin-resistant ovarian cancer cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B67.

Abstract A16: COL11A1 confers cisplatin resistance through fatty acid oxidation in ovarian cancer cells

Abstract Cisplatin resistance is the major challenge in the treatment of ovarian cancer, and it is crucial to develop novel therapeutic strategies to block cisplatin resistance. The purpose of this study was to identify new therapeutic targets for the treatment of cisplatin-resistant ovarian cancer. We previously identified collagen type XI alpha 1 (COL11A1) as a novel biomarker associated with cisplatin resistance in ovarian cancer. COL11A1 is expressed by cancer-associated fibroblasts (CAFs) adjacent to cancer cells as well as cisplatin-resistant ovarian cancer cell lines. Our data indicate that COL11A1 inhibits cisplatin-induced apoptosis in ovarian cancer cells, but not through altered gene expression of many of the well-established mechanisms of cisplatin resistance (i.e., transport, inactivation, DNA repair pathways). Rather, our proteomics data revealed that mitochondrial fatty acid beta-oxidation (FAO) was the most upregulated pathway by COL11A1 in ovarian cancer cells. To determine a link between COL11A1-driven cisplatin resistance and FAO, we knocked down COL11A1 in A2780CIS cisplatin-resistant ovarian cancer cells, a cell line expressing high endogenous levels of COL11A1. COL11A1 knockdown not only decreased the expression of genes involved in fatty acid beta-oxidation, such as CD36, CPT1A, ACAA2, HADHA, but also decreased fatty acid oxidation rate and increased cisplatin-induced apoptosis in A2780CIS ovarian cancer cells. In contrast, ovarian cancer cells became resistant to cisplatin-induced apoptosis and increased fatty acid beta-oxidation when exposed to COL11A1 protein or cocultured with COL11A1-overexpressing CAFs. Importantly, these cancer cells became more sensitive to the FAO inhibitor etomoxir. Collectively, our results suggest that COL11A1 confers cisplatin resistance by increasing FAO in ovarian cancer cells. Our study uncovers FAO as a promising therapeutic target to reduce cisplatin resistance in ovarian cancer, particularly in recurrent ovarian cancers that typically express high levels of COL11A1. Citation Format: Miran Rada, Jennifer Cha, Jessica Sage, Bo Zhou, Wei Yang, Sandra Orsulic, Dong-Joo Cheon. COL11A1 confers cisplatin resistance through fatty acid oxidation in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A16.

Abstract A65: Combination of a thioxodihydroquinazolinone compound with cisplatin eliminates ovarian cancer stem cell-like cells (CSC-LCs) and shows preclinical potential

Abstract Cancer stem cell-like cells (CSC-LCs) contribute to drug resistance and recurrence of ovarian cancer. Strategies that can eradicate CSC-LCs are expected to substantially improve the outcome of ovarian cancer treatments. We have previously identified a class of thioxodihydroquinazolinone small molecules, which have strong synergistic antitumor activity with platinum drugs, the standard chemotherapeutic agents for ovarian cancer treatment. In the current study, using the activity of aldehyde dehydrogenase (ALDH) as a marker of CSC-LCs, we demonstrated that the combination of thioxodihydroquinazolinone compound 19 with cisplatin is able to deplete ALDH-high CSC-LC population in both established platinum-resistant ovarian cancer cell lines and primary ovarian cancer cells from metastatic ascites of a cisplatin-resistant patient. Compound 19 enhanced the accumulation of intracellular cisplatin in ALDH-high ovarian cancer cells. The combination of compound 19 with cisplatin was also able to reduce the sphere-forming capability of cisplatin-resistant ovarian cancer cells. Using a spheroid-based in vitro metastasis model of ovarian cancer, we demonstrated that the combination of compound 19 with cisplatin prevents spheroid ovarian cancer cells from attaching to substratum and spreading. In vivo, the treatment by the combination of compound 19 with cisplatin significantly reduced tumor burden in a cisplatin-resistant intraperitoneal ovarian cancer xenograft mouse model, as compared to cisplatin-alone treatment. Taken together, our study demonstrated that the thioxodihydroquinazolinone compounds are a new class of agents that in combination with cisplatin are capable of eliminating CSC-LCs in ovarian cancer. Further development of thioxodihydroquinazolinone small molecules may yield a more effective treatment for cisplatin-resistant metastatic ovarian cancer. Citation Format: Jing Ma, Joseph Salamoun, Peter Wipf, Robert Edwards, Bennett Van Houten, Wei Qian. Combination of a thioxodihydroquinazolinone compound with cisplatin eliminates ovarian cancer stem cell-like cells (CSC-LCs) and shows preclinical potential. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A65.

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.

Triptolide antagonized the cisplatin resistance in human ovarian cancer cell line A2780/CP70 via hsa-mir-6751.

We aimed to investigate the effect of triptolide on cisplatin resistance in ovarian cancer cell lines. The apoptosis of ovarian cancer cell lines A2780 and A2780/CP70 was determined by flow cytometry. Protein expression levels of Hexokinase 2 (HK2) were detected by western blot. Possible mRNAs which target HK 2 expression were predicted through online tools miRbase. Triptolide-promoted cell apoptosis of cisplatin-resistant ovarian cancer cell line A2780/CP70. Triptolide treatment sensitized the effect of cisplatin in A2780/CP70, as evidenced by decreased survival fraction of A2780/CP70 cells. Mechanically, triptolide treatment inhibited HK 2 expression in A2780/CP70 cells. In addition, Hsa-mir-6751 expression level was upregulated in A2780/CP70 cells after triptolide treatment.

PO-482 Efficacy of inhibition of BAD Ser99 phosphorylation by a novel small molecule in cisplatin resistant ovarian cancer

IntroductionHuman BAD (hBAD) is a pro-apoptotic Bcl-2 family member, whose apoptotic functions can be inactivated through phosphorylation of specific residues including Ser99. Clinically, BAD phosphorylation has been reported to indicate poor survival and cisplatin resistance in ovarian cancer patients. Herein, we investigated the therapeutic potential of inhibiting BAD Ser99 phosphorylation in ovarian cancer.Material and methodsNPB, a novel small molecule which specifically inhibits BAD Ser99 phosphorylation, has been developed in our laboratory. Cell function assays performed include Alamar Blue cell viability assay, caspase3/7 assay, PI-Annexin V apoptosis assay, cell cycle analysis and 3D growth in Matrigel. The CI values are calculated by CompuSyn using Chou-Talalay method. The cancer stem cell-like cell population was examined by ALDEFLOUR and sphere formation assays.Results and discussionsThe level of BAD Ser99 phosphorylation is negatively correlated with cisplatin sensitivity in a panel of ovarian cancer cell lines. BAD Ser99 phosphorylation is increased upon acute cisplatin treatment of both sensitive and resistant ovarian cancer cells. The inhibition of BAD Ser99 phosphorylation by NPB alone increased apoptosis, and decreased cell viability, 3D growth, and anchorage-independent growth of both parental and cisplatin resistant ovarian cancer cells. In particular, NPB increased the sensitivity of the parental cells, and partially re-sensitised cisplatin resistant ovarian cancer cells towards cisplatin, with the combination of NPB and cisplatin showing a synergistic effect. An upstream BAD kinase, AKT has also been reported to mediate cisplatin resistance in ovarian cancer cells. Correspondingly, the combination of NPB and AKT inhibitor AZD5363 exhibited strong synergistic effects in both parental and cisplatin resistant cell lines. We also demonstrated that both the phosphorylation of BAD and its upstream kinase AKT was increased in this CSC-like population. NPB treatment alone was observed to decrease the CSC-like population, while the combination of AZD5363 and NPB produced a synergistic decrease in the CSC-like population.ConclusionOur preclinical data suggests that NPB, as a novel inhibitor of BAD Ser99 phosphorylation, can potentially be used in combination with cisplatin as a therapy for naïve ovarian cancer patients to increase cisplatin sensitivity. Furthermore, the combination of NPB and AKT inhibitor AZD5363 is a potential therapeutic approach in the treatment of cisplatin resistant ovarian cancer.