Cytotoxicity In Ovarian Cancer Cells Research Articles

Enhancing resveratrol pharmacokinetics and cytotoxicity in ovarian cancer cells via nanostructured lipid carriers

Resveratrol (Res), a potent nutraceutical, holds promise in ameliorating cancer and catalyzing breakthroughs in cancer therapy. Despite numerous preclinical studies on its anticancer activity, translational research progress remains limited. Poor pharmacokinetics and low potency are primary bottlenecks of Res therapy. In this study, we aimed to enhance the pharmacokinetic profile of Res in a rat model and assess its cytotoxicity against human ovarian cancer cells by developing Res loaded Nanostructured Lipid Carriers (Res-NLCs). The Res-NLCs were synthesized using the solvent injection method, featuring a size of 177 nm, a PDI of 0.25, and an entrapment efficiency exceeding 80%. The formulation exhibited stability for three months at 4 °C and 40 °C. Cytotoxicity assessments using the MTT assay and cellular uptake studies on human ovarian cancer cells (PA1 and SKOV3 luc) revealed significantly superior time-dependent cytotoxic effects and cellular accumulation of Res-NLCs compared to free Res. Pharmacokinetic studies of intravenously administered Res in NLCs demonstrated prolonged plasma levels of Res for at least 48 h. Remarkably, Res-NLCs exhibited approximately 15- and 5-fold improvements in AUC and elimination half-life (t1/2), respectively, compared to Res in solution in rats. Moreover, Res tissue distribution from Res-NLCs in rats showed significantly higher accumulation in the liver, lungs, and ovaries over 48 h, underscoring the potential for targeted therapy. In conclusion, NLCs effectively mitigated premature reduction of Res in the bloodstream, prolonged circulation period, and enhanced tissue accumulation. These findings suggest promising prospects for improving therapeutic outcomes in ovarian cancer therapy using Res-NLCs.

Daphnetin induces ferroptosis in ovarian cancer by inhibiting NAD(P)H:Quinone oxidoreductase 1 (NQO1)

Ferroptosis, an emerging nonapoptotic, modulated cell death process characterized by iron accumulation and subsequent lipid peroxidation, has been intimately implicated in the development and progression of ovarian cancer (OC). Daphnetin (Daph), a natural product isolated from Daphne Korean Nakai, exhibits anticancer efficacy against various solid tumors. However, the specific role and potential mechanism underlying Daph-mediated modulation of ferroptosis in OC cells remain elusive. This study aims to analyze the proferroptotic impacts of Daph on OC cells and to further explore the underlying mechanisms involved. We used CCK-8, wound healing and Transwell assays to assess whether Daph can inhibit the proliferation and migration of OC cells. Additionally, transmission electron microscopy (TEM), iron measurement, reactive oxygen species (ROS) analysis, lipid peroxidation assays, qRT-PCR and western blotting were utilized to evaluate the impact of Daph on ferroptosis and elucidate the potential underlying mechanism. Furthermore, RNA sequencing analysis, molecular docking analysis, cellular thermal shift assays (CETSAs) and NQO1 activity assays were used to predict and validate the binding and mechanistic interactions between Daph and NQO1. Subcutaneous tumorigenesis models were utilized to examine the effectiveness of Daph (and/or cisplatin) in vivo. Daph exerted antitumor effects by inducing the death and suppressing the migration of A2780 and SKOV3 cells. Further, Daph induced ferroptosis in OC cells, as evidenced by the accumulation of intracellular ferrous iron (Fe2+), ROS and lipid peroxides, as well as the decreases in the glutathione/oxidized glutathione disulfide (GSH/GSSG) ratio and the expression of ferroptosis indicators (SLC7A11 and GPX4). RNA sequencing and molecular docking analyses revealed that the direct interaction between NQO1 and Daph reduced both the activity and expression of NQO1. Importantly, NQO1 overexpression effectively alleviated the effects of Daph on proliferation, migration, and ferroptosis in vitro and in vivo. Interestingly, we also found that combination treatment with Daph, a negative regulator of NQO1, and cisplatin synergistically induced cytotoxicity in OC cells. Our findings are the firstly demonstrated that Daph acts as a novel ferroptosis inducer in OC cells by specifically targeting NQO1 and is thus a promising candidate agent for OC treatment.

Curcumin modulates cell type-specific miRNA networks to induce cytotoxicity in ovarian cancer cells

AimTo understand the epigenetic role of curcumin, a natural polyphenolic compound extracted from the spice Curcuma longa in inducing cytotoxicity in two molecularly distinct ovarian cancer cell lines: PA1 and A2780. Materials and methodsAn integrated mRNA-miRNA sequence analysis was performed to determine the curcumin-induced mRNA–miRNA regulatory networks in the induction of cytotoxicity. The miRNA-mRNA pathways, the miRNAs and their targets implicated in apoptosis, autophagy, DNA damage, and stemness markers were validated. Gene/miRNA expressions were validated using qPCR and protein expressions by western blotting. Curcumin-induced oncogenic /tumor-suppressor miRNAs were profiled utilising the oncomiRdb database. Similarly, the expressions of oncogenes/tumor suppressor genes were profiled and correlated with the TCGA ovarian cancer dataset. A dual luciferase assay was performed to investigate the interaction of miR-199a-5p to its direct target, DDR1. Key findingsThe expression of several miRNAs demonstrated an inverse correlation with their respective direct targets. In curcumin-treated PA1 cells, miR-335-5p target ATG5 (autophagic), and OCT4 (pluripotent gene) were downregulated, miR-32a target PTEN (tumor suppressor) was upregulated, miR-1285 target P53 (tumor suppressor) was upregulated, and both miR-182-5p and miR-503-3p target BCL2, were down-regulated. Contrastingly, in curcumin-treated A2780 cells, miR-181a-3p target ATG5, miR-30a-5p, and miR-216a target BECN1 (autophagic) were upregulated, and miR-129a-5p target BCL2 were downregulated. The reversal of the oncomiR/TSmiR profile revealed suppression of oncogenic processes by curcumin. Curcumin treatment induced a moderate cisplatin-sensitisation effect and impaired epithelial-to-mesenchymal transition (EMT) characteristics. Curcumin also regulated the miR-199a-5p/DDR1 axis with a decrease in collagen deposition. SignificanceThe activity of curcumin is cell-type specific. Distinct miRNA regulatory networks were activated to induce multiple modes of cellular cytotoxicity in these ovarian cancer cells. This study further highlights the molecular mechanism of curcumin action in ovarian cancers establishing its candidacy as a promising drug candidate.

Benzimidazole-Based NHC Metal Complexes as Anticancer Drug Candidates: Gold(I) vs. Platinum(II)

Herein, we present a comparative study on the chemistry and biological activity of N-heterocyclic carbene (NHC)Pt(II)/Au(I) complexes. Accordingly, representative compounds of the cis/trans- [PtL2X2] (X = Cl (5, 6) or I (7, 8)), [PtL3Cl]+ (9), [AuLX] (X = Cl (10) or I (11)), and [AuL2]+ (12) type, where L is 1,3-diethylbenzimidazol-2-ylidene, were synthesized and characterized in detail to elucidate the role of the metal center on their physicochemical and biological properties. The stability of the complexes in the presence of cell culture medium and their reactivity toward relevant biomolecules were investigated by RP-HPLC. In addition, their effects on plasmid DNA and in vitro cytotoxicity in ovarian cancer cells and non-malignant fibroblasts were evaluated. Cationic [AuL2]+ and [PtL3X]+ species displayed the highest cytotoxicity and stability in cell culture medium in the series. They exhibited IC50 values lower than the established metallodrugs cisplatin and auranofin in both wild-type and cisplatin-resistant ovarian cancer cells, being able to circumvent cisplatin resistance. Finally, Pt(II)–NHC complexes form 5′-guanosine monophosphate adducts under physiologically relevant conditions and interact with plasmid DNA in contrast to their Au(I) analogs, corroborating their distinct modes of action.

A Novel Antibody-Drug Conjugate Targeting Nectin-2 Suppresses Ovarian Cancer Progression in Mouse Xenograft Models.

Ovarian cancer is the fifth leading cause of cancer, followed by front line is mostly platinum agents and PARP inhibitors, and very limited option in later lines. Therefore, there is a need for alternative therapeutic options. Nectin-2, which is overexpressed in ovarian cancer, is a known immune checkpoint that deregulates immune cell function. In this study, we generated a novel anti-nectin-2 antibody (chimeric 12G1, c12G1), and further characterized it using epitope mapping, enzyme-linked immunosorbent assay, surface plasmon resonance, fluorescence-activated cell sorting, and internalization assays. The c12G1 antibody specifically bound to the C2 domain of human nectin-2 with high affinity (KD = 2.90 × 10−10 M), but not to mouse nectin-2. We then generated an antibody-drug conjugate comprising the c12G1 antibody conjugated to DM1 and investigated its cytotoxic effects against cancer cells in vitro and in vivo. c12G1-DM1 induced cell cycle arrest at the mitotic phase in nectin-2-positive ovarian cancer cells, but not in nectin-2-negative cancer cells. c12G1-DM1 induced ~100-fold cytotoxicity in ovarian cancer cells, with an IC50 in the range of 0.1 nM~7.4 nM, compared to normal IgG-DM1. In addition, c12G1-DM1 showed ~91% tumor growth inhibition in mouse xenograft models transplanted with OV-90 cells. These results suggest that c12G1-DM1 could be used as a potential therapeutic agent against nectin-2-positive ovarian cancers.

FBP1 knockdown decreases ovarian cancer formation and cisplatin resistance through EZH2-mediated H3K27me3.

Worldwide, ovarian cancer (OC) is the seventh common cancer and the second most common cause of cancer death in women. Due to high rates of relapse, there is an urgent need for the identification of new targets for OC treatment. The far-upstream element binding protein 1 (FBP1) and enhancer of zeste homolog 2 (EZH2) are emerging proto-oncogenes that regulate cell proliferation and metastasis. In the present study, Oncomine data analysis demonstrated that FBP1 was closely associated with the development of OC, and The Cancer Genome Atlas (TCGA) data analysis indicated that there was a positive correlation between FBP1 and EZH2 in ovarian tissues. Moreover, we found that FBP1 knockdown suppressed tumor formation in nude mice and cisplatin resistance of OC cells, but the role of FBP1 in the cisplatin resistance of OC cells remained unclear. In addition, we verified physical binding between FBP1 and EZH2 in OC cells, and we demonstrated that FBP1 knockdown enhanced cisplatin cytotoxicity in OC cells and down-regulated EZH2 expression and trimethylation of H3K27. These results suggested that FBP1 increases cisplatin resistance of OC cells by up-regulating EZH2/H3K27me3. Thus, FBP1 is a prospective novel target for the development of OC treatment.

Enhancement of tumor tropism of mPEGylated nanoparticles by anti-mPEG bispecific antibody for ovarian cancer therapy

Ovarian cancer is highly metastatic, with a high frequency of relapse, and is the most fatal gynecologic malignancy in women worldwide. It is important to elevate the drug susceptibility and cytotoxicity of ovarian cancer cells, thereby eliminating resident cancer cells for more effective therapeutic efficacy. Here, we developed a bispecific antibody (BsAb; mPEG × HER2) that can easily provide HER2+ tumor tropism to mPEGylated liposomal doxorubicin (PLD) and further increase the drug accumulation in cancer cells via receptor-mediated endocytosis, and improve the cytotoxicity and therapeutic efficacy of HER2+ ovarian tumors. The mPEG × HER2 can simultaneously bind to mPEG molecules on the surface of PLD and HER2 antigen on the surface of ovarian cancer cells. Simply mixing the mPEG × HER2 with PLD was able to confer HER2 specificity of PLD to HER2+ ovarian cancer cells and efficiently trigger endocytosis and enhance cytotoxicity by 5.4-fold as compared to non-targeted PLD. mPEG × HER2-modified PLD was able to significantly increase the targeting and accumulation of HER2+ ovarian tumor by 220% as compared with non-targeted PLD. It could also significantly improve the anti-tumor activity of PLD (P < 0.05) with minimal obvious toxicity in a tumor-bearing mouse model. We believe that the mPEG × HER2 can significantly improve the therapeutic efficacy, potentially reduce the relapse freqency and thereby achieve good prognosis in ovarian cancer patients.

Siomycin A induces reactive oxygen species-mediated cytotoxicity in ovarian cancer cells.

Ovarian cancer is one of the leading causes of cancer-related death among women worldwide and accounts for 4% of all cancer cases in female patients. To date, ovarian cancer has the poorest prognosis among all types of gynecological cancer; thus, it is necessary to identify prospective therapeutic options. Previous studies have demonstrated the involvement of reactive oxygen species (ROS) in the cytotoxicity of various anticancer drugs against several types of carcinoma, including ovarian cancer. The present study aimed to investigate the anticancer effects of Siomycin A, a thiopeptide antibiotic, on the ovarian cancer cell lines PA1 and OVCAR3. To determine the viability of these cells following exposure to Siomycin A, the MTT assay was used, and apoptosis was determined by ELISA. In addition, mitochondrial membrane potential was determined by JC1 staining, and cellular ROS levels were assessed by dichlorodihydrofluorescein diacetate staining in the presence and absence of antioxidant NAC. The subsequent levels of antioxidant enzymes and glutathione were also determined following Siomycin A treatment in the two cell lines. A combination study with Siomycin A and cisplatin indicated enhanced efficiency of the drugs on ovarian cancer cell viability. The results of the present study also demonstrated that Siomycin A induced ROS production, inhibited the major antioxidant enzymes, including catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and intracellular GSH in PA1 and OVCAR3 cells, and inhibited the cell viability with an IC50 of ~5.0 and 2.5 µM after 72 h respectively compared with the untreated controls. Additionally, the Siomycin A-induced ROS production further targeted apoptotic cell death by impairing the mitochondrial membrane potential and modulating the levels of pro- and antiapoptotic proteins compared with those in the corresponding control groups. The administration of the antioxidant N-acetylcysteine significantly abrogated the cytotoxic effects of Siomycin A. In conclusion, the results of the present study demonstrated the role of ROS in Siomycin A-mediated cytotoxicity in ovarian cancer cells.

Supramolecular self-associating amphiphiles (SSAs) as nanoscale enhancers of cisplatin anticancer activity.

Many chemotherapeutic drugs have a narrow therapeutic window due to inefficient tumour cell permeation. Supramolecular self-associating amphiphilic salts (SSAs) are a unique class of small molecules that offer potential as next generation cancer drugs and/or therapeutic enhancement agents. Herein, we demonstrate the cytotoxicity of seven SSAs towards both ovarian and glioblastoma cancer cells. We also utilize the intrinsic fluorescent properties of one of these lead SSAs to provide evidence for this class of compound to both bind to the exterior cancer cell surface and permeate the cell membrane, to become internalized. Furthermore, we demonstrate synergistic effects of two lead SSAs on cisplatin-mediated cytotoxicity of ovarian cancer cells and show that this correlates with increased DNA damage and apoptosis versus either agent alone. This work provides the first evidence that SSAs interact with and permeate cancer cell membranes and enhance the cytotoxic activity of a chemotherapeutic drug in human cancer cells.

MAP30 protein from Momordica charantia is therapeutic and has synergic activity with cisplatin against ovarian cancer in vivo by altering metabolism and inducing ferroptosis

Increasing evidence shows that Traditional Chinese Medicine (TCM) has an obvious appeal for cancer treatment, but there is still a lack of scientific investigation of its underlying molecular mechanisms. Bitter melon or bitter gourd (Momordica charantia) is an edible fruit that is commonly consumed, and it is used to cure different diseases in various ancient folk medical practices. We report that a bioactive protein, MAP30, isolated from bitter melon seeds exhibited potent anticancer and anti-chemoresistant effects on ovarian cancer cells. Functional studies revealed that MAP30 inhibited cancer cell migration, cell invasion, and cell proliferation in various ovarian cancer cells but not normal immortalized ovarian epithelial cells. When administered with cisplatin, MAP30 produced a synergistic effect on cisplatin-induced cell cytotoxicity in ovarian cancer cells. When low doses of cisplatin and MAP30 were co-injected intraperitoneally, a remarkable reduction of tumor dissemination and tumor growth was observed in an ovarian cancer ascites mouse model. Notably, blood tests confirmed that MAP30 did not cause any adverse effects on liver and kidney functions in the treated mice. MAP30 activated AMP-activated protein kinase (AMPK) signaling via CaMKKβ and induced cell cycle arrest in the S-phase. MAP30 modulated cell metabolism of ovarian cancer cells via suppression of GLUT-1/-3-mediated glucose uptake, adipogenesis, and lipid droplet formation in tumor development and progression. MAP30 also induced an increase in intracellular Ca2+ ion concentration, which triggered ROS-mediated cancer cell death via apoptosis and ferroptosis. Collectively, these findings suggest that natural MAP30 is a non-toxic supplement that may enhance chemotherapeutic outcomes and benefit ovarian cancer patients with peritoneal metastases.

Abstract 6593: PRGN-3005 UltraCAR-T™: multigenic CAR-T cells generated using non-viral gene delivery and rapid manufacturing process for the treatment of ovarian cancer

Abstract Ovarian cancer is the most lethal of gynecologic cancers. Chimeric antigen receptor (CAR)-T cells have improved clinical outcomes in hematological malignancies; however similar responses have not yet been achieved for solid tumors. Furthermore, traditional methods for CAR-T manufacturing utilize viral vectors and weeks of ex vivo expansion to achieve clinically relevant cell numbers. This leads to CAR-T cells with an exhausted T cell phenotype, high manufacturing costs, and treatment delays. We have developed the UltraCAR-T™ platform to overcome these limitations by utilizing an advanced non-viral gene delivery system and a rapid, decentralized manufacturing process. Our non-viral gene delivery platform is based on Sleeping Beauty transposon system precision engineered for optimal delivery of multiple genes of UltraCAR-T. UltraCAR-T cells are manufactured at medical center's cGMP facility using autologous T cells and administered back to patient only one day after gene transfer. PRGN-3005 UltraCAR-T cells simultaneously co-express (i) a CAR specifically targeting MUC16 which is highly expressed on ovarian tumors with limited normal tissue expression; (ii) membrane bound IL-15 (mbIL15) for enhanced in vivo expansion and persistence; and (iii) a kill switch for improved therapeutic control. Healthy donor T cells were transfected with non-viral system encoding CAR, mbIL15 and kill switch to generate PRGN-3005 cells. Expression of transgenes was confirmed by flow cytometry and western blot analyses. PRGN-3005 demonstrated a robust MUC16-specific cytotoxicity of ovarian cancer cells in vitro. PRGN-3005 exhibited a stem-cell like memory phenotype, significant improvement in survival even in absence of exogenous cytokines and lacked autonomous proliferation in vitro in the absence of MUC16 compared to conventional CAR-T cells, demonstrating beneficial effects of mbIL15 on phenotype and persistence of UltraCAR-T cells. Furthermore, we demonstrated that PRGN-3005 cells were selectively and effectively eliminated by kill switch activator antibody via ADCC. For in vivo anti-tumor efficacy of PRGN-3005, an ovarian cancer model of MUC16+ SK-OV-3 cell line was evaluated in NSG mice. In tumor-bearing mice, a single administration of PRGN-3005 one day after gene transfer resulted rapid in vivo expansion and significant increase in UltraCAR-T cells, in particular T stem cell memory (TSCM) population compared to mbIL15neg CAR-T cells. PRGN-3005 demonstrated significantly higher anti-tumor effect (p&amp;lt;0.01) compared to mbIL15neg CAR-T and led to complete tumor regression in all treated mice. PRGN-3005 UltraCAR-T administered one day after non-viral gene transfer has superior anti-tumor efficacy and persistence compared traditional CAR-T cells and represent a promising opportunity for ovarian cancer treatment. Based on these results, FDA has approved an IND application and the first in human PRGN-3005 Phase I clinical trial for advanced ovarian cancer is currently under evaluation (NCT03907527). Citation Format: Tim Chan, Marion Chakiath, Lindsey Shepard, Simon Metenou, Fernando Carvajal-Borda, Jose Velez, Adeline Govekung, Jacques Plummer, Carol Poortman, Padma Kamineny, Xiaohong Ma, Rutul R. Shah, Mary L. Disis, Helen Sabzevari. PRGN-3005 UltraCAR-T™: multigenic CAR-T cells generated using non-viral gene delivery and rapid manufacturing process for the treatment of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6593.

Synergistic enhancement of efficacy of platinum drugs with verteporfin in ovarian cancer cells

BackgroundEpithelial ovarian cancers (EOCs) comprises the majority of malignant ovarian neoplasms. Combination treatment with chemotherapeutic agents seems to be a promising strategy in ovarian cancer (OVCA) patients in order to overcome drug resistance. In this in vitro study, we investigated the therapeutic efficacy of verteporfin (VP) alone and in combination with cisplatin (CDDP), carboplatin (CP) and paclitaxel (Taxol). The main objectives of this study are to determine the nature of interactions between VP and CDDP/CP/Taxol and to understand the mechanism of action of VP in OVCA cells.MethodsThe efficacy of VP on cell proliferation, cytotoxicity, invasion and clonogenic capacity was assayed in CDDP-sensitive (COV504, OV-90) and CDDP-resistant (A2780Cis) cell lines. The cytotoxic effects of drugs either alone or in combination were evaluated using MTT assay and Cell Viability Blue assay. The effects of drugs on the metabolic functions were studied using matrigel invasion assay and clonogenic assay. Immunoblot analysis was carried out to investigate changes in YAP and cell cycle genes. Changes in the cytokines due to drug treatments were analyzed using a cytokine array.ResultsTreatment with VP inhibited cell proliferation, invasion and increased cytotoxicity of OVCA cells. We observed that VP chemosensitized CDDP-resistant cells, even at lower doses. When added either in constant or non-constant ratios, VP produced synergistic effects in combination with CDDP/CP/Taxol. A cytokine array identified upregulation of cytokines in OVCA cells that were inhibited by VP treatment.ConclusionsEither in cisplatin-resistant cell lines or cisplatin-sensitive cell lines, VP proves to be more efficient in inhibiting cell proliferation and inducing cytotoxicity. Our results suggest that novel combinations of VP with CDDP or CP or Taxol might be an attractive therapeutic strategy to enhance OVCA chemosensitivity. The fact that lower doses of VP are effective in chemosensitizing the CDDP-resistant cells, might ultimately lead to the development of an innovative combination therapy for the treatment of OVCA patients.

Gold Nanoparticles as anti-ovarian cancer therapy

Cancer of the ovary is one of the most common types of women cancer in the world. In Iraq, it is seventh cancer in Iraqi women. Nanomedicine emerged as a possible alternative for cancer therapy. Many nanoparticles have been tested on different cancer cell lines with promising results. Gold nanoparticles (AuNPs) showed interesting biocompatibility properties such as selective accumulation in tumor cells. The currentwork was aimed to study cytotoxicity and cell death induced by AuNPson ovarian cancer cells. Methods: Cytotoxicity of AuNPs was assessed by MTT viability assay. Results: AuNPs nanoparticles from 25 up to 100 µg/mL for 72 hours exerted concentration-dependent cytotoxicity. Conclusion: Gold nanoparticles induce cytotoxicity in ovarian cancer cells and Induce cell death and considered promising as cancer therapy.

CircCELSR1 (hsa_circ_0063809) Contributes to Paclitaxel Resistance of Ovarian Cancer Cells by Regulating FOXR2 Expression via miR-1252

Ovarian cancer is the malignant tumor of the female reproductive system with the highest fatality rate. Tolerance to chemotherapeutic drugs such as paclitaxel (PTX) occurring in the very early stage is one of the important factors of the poor prognosis of ovarian cancer. Herein, we aim to study the dysregulation of a particular circular RNA (circRNA), circCELSR1 (hsa_circ_0063809), and its role in the progression and PTX resistance of ovarian cancer. The high expression of circCELSR1 in PTX-resistant tissues of ovarian cancer and PTX-resistant ovarian cancer cells (SKOV3/PTX and HeyA-8/PTX) was determined by microarray analyses and quantitative real-time PCR. Cell Counting Kit-8 (CCK-8) assays were performed to investigate the effect of circCELSR1 on PTX sensitivity of ovarian cancer cells. Flow cytometer assays were used to detect cell cycle and apoptosis of ovarian cancer cells. The effect of circCELSR1 on ovarian cancer cells was assessed in vitro and in vivo. The microRNA (miRNA) sponge mechanism of circRNAs was demonstrated using dual-luciferase reporter and RNA immunoprecipitation assays. By microarray (5 PTX-resistant ovarian cancer tissues νs 5 PTX-sensitive ovarian cancer tissues) and qRT-PCR (36 normal ovarian tissues and ovarian cancer tissues) we identified circCELSR1 to be dramatically highly expressed in ovarian cancer samples and correlated with PTX resistance. Compared with sensitive cell lines, circCELSR1 was also highly expressed in PTX-resistant ovarian cancer cell lines, and circCELSR1 silencing enhanced PTX-induced cytotoxicity in ovarian cancer cells. Meanwhile, the inhibition of circCELSR1 also caused ovarian cancer cell G0/G1 arrest and an increase in apoptosis. In vivo studies revealed that circCELSR1 was stably inhibited in a xenograft mouse model and inhibited the growth of ovarian cancer. Furthermore, we demonstrated that circCELSR1 acts as a sponge for miR-1252 and verified that forkhead box 2 (FOXR2) is a novel target of miR-1252. In this study, we explored the specific mechanisms of PTX resistance and tumor progress of ovarian cancer due to circCELSR1; presented the circCELSR1-miR-1252-FOXR2 axis and its role in ovarian cancer drug sensitivity and progression; and suggest that the results may provide an experimental basis for clinical application.

Abstract NT-104: SYNERGISTIC COMBINATION OF MORTALIN-TARGETING AND P53 REACTIVATOR DRUGS FOR OVARIAN CANCER

Abstract PURPOSE: TP53 gene missense mutations represent the most common genetic defect in fallopian tube (FT) lesions and high grade serous ovarian cancer (HGSOC) and cause of drug resistance. We hypothesized that drugs with complementary p53 protein-targeted mechanisms (SHetA2 drug release of p53 from mortalin binding, PRIMA-1MET drug reactivation of p53 transcription activity) synergistically kill ovarian cancer cells without harming normal cells. EXPERIMENTAL DESIGN: SHetA2-mediated cytoplasmic to nuclear/mitochondrial p53 translocation was studied in wildtype and mutant p53 ovarian cancer cell lines using Western blot analysis of subcellular-enriched fractions and fluorescent microscopic imaging of immunocytochemistry. Human FT epithelial cells (hFTSECs) were used as healthy controls. The p53 reactivator drugs PRIMA-1 and PRIMA-1MET were combined with SHetA2 to study drug interactions and mechanisms using Western blot, rtPCR, cytotoxicity, reactive oxygen species (ROS) and ATP assays. RESULTS: SHetA2 induced p53 nuclear and mitochondrial accumulation in ovarian cancer cell lines, but not in hFTSECs. Both mortalin and p53 expression altered SHetA2 cytotoxicity in ovarian cancer cells. Modest effects of PRIMA-1MET on cell lines inversely correlated with SLC7A11 expression, a known biomarker of PRIMA-1MET sensitivity. SHetA2 induced SLC7A11 in the presence of wildtype or missense mutant p53, but inhibited SLC7A11 in the absence of p53. SHetA2 and PRIMA-1MET were synergistic-to-strongly synergistic in ovarian cancer cells with mutant p53, synergistic with wildtype p53, additive with null p53 and antagonistic in hFTSECs. Elevated apoptosis, p53 transcription activity and ROS in combination with reduced ATP were associated with this mutant p53-dependent synergy. CONCLUSION: Altogether, our findings identified the SHetA2 and PRIMA-1MET combination as a promising new therapeutic strategy for the treatment of HGSOCs with missense mutant p53. GRANT SUPPORT: Supported by the US National Cancer Institute grant R01 CA196200 and Foundation for Women's Cancer- Ovarinnovate: Ovarcome Research Excellence Award. Citation Format: Satish Kumar Ramraj, Doris M. Benbrook. SYNERGISTIC COMBINATION OF MORTALIN-TARGETING AND P53 REACTIVATOR DRUGS FOR OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-104.

H-Ferritin Affects Cisplatin-Induced Cytotoxicity in Ovarian Cancer Cells through the Modulation of ROS.

Reactive oxygen species (ROS) mediates cisplatin-induced cytotoxicity in tumor cells. However, when cisplatin-induced ROS do not reach cytotoxic levels, cancer cells may develop chemoresistance. This phenomenon can be attributed to the inherited high expression of antioxidant protein network. H-Ferritin is an important member of the antioxidant system due to its ability to store iron in a nontoxic form. Altered expression of H-Ferritin has been described in ovarian cancers; however, its functional role in cisplatin-based chemoresistance of this cancer type has never been explored. Here, we investigated whether the modulation of H-Ferritin might affect cisplatin-induced cytotoxicity in ovarian cancer cells. First, we characterized OVCAR3 and OVCAR8 cells for their relative ROS and H-Ferritin baseline amounts. OVCAR3 exhibited lower ROS levels compared to OVCAR8 and greater expression of H-Ferritin. In addition, OVCAR3 showed pronounced growth potential and survival accompanied by the strong activation of pERK/pAKT and overexpression of c-Myc and cyclin E1. When exposed to different concentrations of cisplatin, OVCAR3 were less sensitive than OVCAR8. At the lowest concentration of cisplatin (6 μM), OVCAR8 underwent a consistent apoptosis along with a downregulation of H-Ferritin and a consistent increase of ROS levels; on the other hand, OVCAR3 cells were totally unresponsive, H-Ferritin was almost unaffected, and ROS amounts met a slight increase. Thus, we assessed whether the modulation of H-Ferritin levels was able to affect the cisplatin-mediated cytotoxicity in both the cell lines. H-Ferritin knockdown strengthened cisplatin-mediated ROS increase and significantly restored sensitivity to 6 μM cisplatin in resistant OVCAR3 cells. Conversely, forced overexpression of H-Ferritin significantly suppressed the cisplatin-mediated elevation of intracellular ROS subsequently leading to a reduced responsiveness in OVCAR8 cells. Overall, our findings suggest that H-Ferritin might be a key protein in cisplatin-based chemoresistance and that its inhibition may represent a potential approach for enhancing cisplatin sensitivity of resistant ovarian cancer cells.

Abstract 5927: Assessing the efficacy of a novel gene therapy approach for treating ovarian cancer: Combination RRV-mediated prodrug activated suicide gene therapy

Abstract Introduction/Aim: Retroviral replicating vectors (RRVs) afford a unique strategy for gene therapy targeting cancer cells. By “activating” RRVs with prodrug activator suicide genes, these RRVs become strong gene delivery vehicles that replicate selectively in cancer cells, and stably persist through proviral genomic integration, leading to inducible cell death upon prodrug administration. This strategy has shown highly promising clinical results in early phase trials (published), and is currently being evaluated in an international Phase III clinical trial for patients with recurrent high-grade glioma. Recently, we have also applied RRV-mediated gene therapy to preclinical models of ovarian cancer. The aim of this current study was to evaluate feasibility and efficacy of combination suicide gene therapy using two RRVs encoated (‘pseudotyped') with different envelope proteins and delivering different prodrug activators for the treatment of ovarian cancer. Methods: Viral spread was monitored in established and primary patient-derived ovarian cancer cell lines (SKOV3, OCI-P5X, OCI-C5X) over the course of ≥27 days using amphotropic murine leukemia virus (AMLV) envelope- and gibbon ape leukemia virus (GALV) envelope-pseudotyped RRVs delivering either fluorescent protein reporter genes (GFP, mStrawberry) or prodrug activator genes (yeast cytosine deaminase, E. coli nitroreductase), either individually or in combination, at various multiplicities of infection (MOI). Flow cytometry for GFP, mStrawberry, and Gag viral protein was used to determine transduction levels and transgene expression levels of RRVs at serial time points. Each therapeutic RRV was tested by MTS assay for its ability to induce cytotoxicity upon exposure to different concentrations prodrug. To determine viral integration and vector stability, PCR of genomic DNA extracted from infected cells was performed using RRV specific primers, and qRT-PCR was performed to determine vector copy number per cell. Results: All ovarian cancer cell lines tested could be &amp;gt;80% transduced by AMLV- and GALV-pseudotyped RRVs, both individually and in combination, within 6-24 days even after initial inoculation at MOI 0.001, with replication kinetics varying between different cell lines. Both RRVs could stably integrate into the target cell genome without evidence of transgene deletion. MTS assays demonstrated that prodrug activated cytotoxicity led to efficient killing of RRV-transduced ovarian cancer cells in vitro, with synergistic cytotoxicity observed at lower prodrug concentrations after combined treatment. Conclusion: RRV-mediated prodrug activator gene therapy may be an effective approach for efficiently and selectively inducing cytotoxicity in ovarian cancer cells. These data support further evaluation of this strategy through translational studies in vivo. Citation Format: James Grosso, Sara Collins, Aki Inagaki, Brian Slomovitz, Noriyuki Kasahara. Assessing the efficacy of a novel gene therapy approach for treating ovarian cancer: Combination RRV-mediated prodrug activated suicide gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5927.

Tuning microtubule dynamics to enhance cancer therapy by modulating FER-mediated CRMP2 phosphorylation

Though used widely in cancer therapy, paclitaxel only elicits a response in a fraction of patients. A strong determinant of paclitaxel tumor response is the state of microtubule dynamic instability. However, whether the manipulation of this physiological process can be controlled to enhance paclitaxel response has not been tested. Here, we show a previously unrecognized role of the microtubule-associated protein CRMP2 in inducing microtubule bundling through its carboxy terminus. This activity is significantly decreased when the FER tyrosine kinase phosphorylates CRMP2 at Y479 and Y499. The crystal structures of wild-type CRMP2 and CRMP2-Y479E reveal how mimicking phosphorylation prevents tetramerization of CRMP2. Depletion of FER or reducing its catalytic activity using sub-therapeutic doses of inhibitors increases paclitaxel-induced microtubule stability and cytotoxicity in ovarian cancer cells and in vivo. This work provides a rationale for inhibiting FER-mediated CRMP2 phosphorylation to enhance paclitaxel on-target activity for cancer therapy.

Novel expression of CD11b in epithelial ovarian cancer: Potential therapeutic target

ObjectiveThe objective of this study was to determine the expression, and effect of targeting CD11b with a monoclonal antibody in ovarian cancer cells. MethodsCD11b expression was determined in epithelial ovarian cancer (EOC) cell lines and tissues by immunofluorescence and flow cytometry. Cytotoxicity of the CD11b antibody and synergism with chemothearapeutic drugs were determined by the MTT Cell Proliferation Assay in human macrophages, normal ovarian epithelial cells, and in both sensitive and chemoresistant EOC cell lines. Cell migration was assessed with a scratch assay and in vivo effects of the CD11b antibody was assessed with a nude mouse ovarian cancer xenograft model. Data was analyzed with either t-tests or one-way ANOVA. ResultsCD11b was unexpectedly expressed in several EOC lines and tissues, but not normal tissues. Targeting CD11b with its monoclonal antibody resulted in intriguing cytotoxic effects in sensitive and chemoresistant EOC lines, while surprisingly not affecting normal cells. More importantly, the cytotoxicity of the CD11b antibody when combined with chemotherapeutic drugs (cisplatin or docetaxel) was significantly synergistic, in both sensitive and chemoresistant EOC cells. The anti-tumorigenic effect of the CD11b antibody was confirmed in an ovarian cancer nude mouse xenograft model. ConclusionHere we identify CD11b as a novel target, which selectively induces cytotoxicity in ovarian cancer cells.

Polyethylene glycol modified ORMOSIL theranostic nanoparticles for triggered doxorubicin release and deep drug delivery into ovarian cancer spheroids

A novel pegylated multifunctional probe of Ormosil nanoparticles (PEGCDSIR820) loaded with Near Infrared dye (NIR; IR820) and a chemotherapeutic drug, Doxorubicin (DOX) was developed for cancer theranostic applications. PEGCDSIR820 nanoparticles had an average diameter of 58.2±3.1nm, zeta potential of −6.9±0.1mV in cell culture media and stability against aggregation in physiological buffers. The encapsulation efficiency of DOX was 65.0±3.0%, and that of IR820 was 76.0±2.1%. PEGCDSIR820 showed no cytotoxicity in ovarian cancer cells (Skov-3). The cytotoxicity markedly increased when Skov-3 cells incubated with PEGCDSIR820 particles were exposed to 808nm laser due to the combination of adjuvant hyperthermia (43°C) and enhanced DOX release. Exposure to laser enhanced the release of DOX, 45% of DOX release was observed in 3h compared to 23% without laser exposure. Confocal imaging in Skov-3 cells showed that the combination of hyperthermia due to NIR exposure and release of DOX caused cell necrosis. Furthermore, in spheroids exposed to NIR laser penetration of DOX was deeper compared to the absence of laser exposure. Skov-3 spheroids incubated with pegylated nanoparticles for 24h and exposed to laser showed 94% reduction in cell viability. Encapsulation of IR820 in PEGCDSIR820 increased the in-vivo elimination half-life to 41.0±7.2h from 30.5±0.5h of free IR820.