Ovarian Cancer Cell Lines OVCAR3 Research Articles

Metabolomics profiling and chemoresistance mechanisms in ovarian cancer cell lines: Implications for targeting glutathione pathway

Ovarian cancer presents a significant challenge due to its high rate of chemoresistance, which complicates the effectiveness of drug-response therapy. This study provides a comprehensive metabolomic analysis of ovarian cancer cell lines OVCAR-3 and SK-OV-3, characterizing their distinct metabolic landscapes. Metabolomics coupled with chemometric analysis enabled us to discriminate between the metabolic profiles of these two cell lines. The OVCAR-3 cells, which are sensitive to doxorubicin (DOX), exhibited a preference for biosynthetic pathways associated with cell proliferation. Conversely, DOX-resistant SK-OV-3 cells favored fatty acid oxidation for energy maintenance. Notably, a marked difference in glutathione (GSH) metabolism was observed between these cell lines. Our investigations further revealed that GSH depletion led to a profound change in drug sensitivity, inducing a shift from a cytostatic to a cytotoxic response. The results derived from this comprehensive metabolomic analysis offer potential targets for novel therapeutic strategies to overcome drug resistance. Our study suggests that targeting the GSH pathway could potentially enhance chemotherapy's efficacy in treating ovarian cancer.

A novel flavonol glycoside and six derivatives of quercetin and kaempferol from Clematis flammula with antioxidant and anticancer potentials

Clematis flammula leaves are traditionally used in Algeria to treat rheumatoid arthritis. Our aim was to identify the main compounds in this plant in order to characterize its antioxidant and anticancer activities.A new flavonol compound, kaempferol 3-O-[(6-O- caffeoyl)- glucosyl(1 → 2)]-(6-Ocaffeoyl) glucoside-7-O-rhamnoside (6) along with six known flavonol molecules were isolated from an ethanolic extract of Clematis flammula leaves. The chemical structures of these flavonols were elucidated using NMR and high resolution-MS spectroscopies. Antioxidant activities of the extract were revealed through its elimination of superoxide radical (O2.-) produced enzymatically (49.7 ± 1.52% at 50 μg/ml) and non-enzymatically (34 ± 1.2% at 100 μg/ml), probably related to its inhibition of the xanthine oxidase form of the xanthine oxidoreductase (XOR) enzyme (25.05 ± 2.33 μg/mL at 100 μg/mL), but mostly to that of the NADH oxidase form of the enzyme (69.16 ± 4.0%). Cytotoxicity tests of the extract on human hepatoma cell line HepG2 and ovarian cancer cell lines A2780 and OVCAR3 were promising especially regarding A2780 cell line (IC50: 77.0 μg/mL), which was comparable to taxol (IC50:76.9 μg/mL).

Tumor sensitization to PARP inhibitors by DNA damaging synthetically lethal acylfulvenes.

e15123 Background: 20-30% of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor some level of deficiency in DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DNA damaging agents. The concept of synthetic lethality has been leveraged to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful in Homologous Recombination Deficiency (HRD) cancers. LP-100 and LP-184 are acylfulvene (AF) class small molecule prodrugs that are metabolized to active compounds by prostaglandin reductase 1 (PTGR1) that is often elevated in solid tumors. AF-induced DNA lesions are likely selectively processed by repair pathways including BRCA1/2 mediated Homologous Recombination (HR). Methods: Cell Titer Glo based 10 day cell viability assays were used to generate drug IC50 values in vitro. Bliss synergy scores were computed with scores ≥ 10 reflecting synergy. Drug-induced DNA double strand breaks (DSB) were detected and quantified with STRIDE (SensiTive Recognition of Individual DNA Ends) assay in cancer cells. A subcutaneous tumor xenograft model was used to determine tumor volumes in control and drug treated mice. Results: In a BRCA2 mutant ovarian cancer cell line OVCAR3, LP-184 and olaparib co-treatment in vitro showed synergistic effects (Bliss score 10). Similarly in a BRCA2 mutant prostate cancer cell line 22Rv1, LP-100 combination with rucaparib was synergistic (Bliss score 18). 400 nM LP-184 showed a time dependent increase in DNA DSB signals in vitro, 1.7X more so in BRCA2 mutant vs wild type (WT) DLD-1 isogenic colon cancer cell lines up to 24 hours. To validate the hypothesis that AF-induced DSBs remain unresolved if exposed to and further synergize with PARPi in vivo, we tested LP-100 plus olaparib in a DU145 (BRCA2 mutant) prostate cancer xenograft model. LP-100 + olaparib treatment resulted in sustained, durable tumor regression indicating in vivo synergy whereas either agent alone did not produce tumor shrinkage. Day 42 tumor growth inhibition was 66% for 2.5 mg/kg LP-100 alone, 34% for 40 mg/kg olaparib alone and 108% for the combination. Moreover, LP-184 treatment resulted in early onset and complete tumor regression in 7 PDX models of PARPi resistant triple negative breast cancers (TNBC) harboring BRCA1/2 loss whereas olaparib/ niraparib remained entirely ineffective in these. Conclusions: AF response is influenced by tumor DNA repair competence. LP-100 and LP-184 are synthetically lethal resulting from their abilities to cause unresolvable DNA damage where tumor cells express high PTGR1 and are deficient in the HR pathway. LP-184 single agent was superior to PARPi in TNBC and LP-100 combination made an olaparib resistant tumor highly sensitive, thereby facilitating a path to extend the opportunities for both AFs and PARPis and enhance the clinical utilization of PARPi.

Apatinib combined with olaparib induces ferroptosis via a p53-dependent manner in ovarian cancer.

PARP inhibitors combined with antiangiogenic drugs have been reported to improve outcomes in BRCA wild-type ovarian cancer patients, the mechanism of the combination is unclear. In this study, we explored the mechanism of apatinib combined with olaparib in the treatment of ovarian cancer. In this study, human ovarian cancer cell lines A2780 and OVCAR3 were used as experimental objects, and the expression of ferroptosis-related protein GPX4 after treatment with apatinib and olaparib was detected by Western blot. The SuperPred database was used to predict the target of the combined action of apatinib and olaparib, and the predicted results were verified by Western blot experiment to explore the mechanism of ferroptosis induced by apatinib and olaparib. Apatinib combined with olaparib-induced ferroptosis in p53 wild-type cells, and p53 mutant cells developed drug resistance. The p53 activator RITA sensitized drug-resistant cells to ferroptosis induced by apatinib combined with olaparib. Apatinib combined with olaparib-induced ferroptosis via a p53-dependent manner in ovarian cancer. Further studies showed that apatinib combined with olaparib-induced ferroptosis by inhibiting the expression of Nrf2 and autophagy, thereby inhibiting the expression of GPX4. The Nrf2 activator RTA408 and the autophagy activator rapamycin rescued the combination drug-induced ferroptosis. This discovery revealed the specific mechanism of ferroptosis induced by apatinib combined with olaparib in p53 wild-type ovarian cancer cells and provided a theoretical basis for the clinical combined use of apatinib and olaparib in p53 wild-type ovarian cancer patients.

Abstract 6241: LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in DNA damage repair deficient cancers

Abstract As many as one third of solid tumors (ovarian, breast, pancreatic, prostate cancers) harbor deficiency in some DNA Damage Repair (DDR) pathways. Such cancers become dependent on alternative DDR pathways to maintain genome integrity, making them selectively vulnerable to DDR inhibitors. The concept of synthetic lethality has been successfully applied to develop strategies to treat subsets of such tumors. Clinically, PARP inhibitors (PARPi) have been successful particularly in Homologous Recombination Deficiency (HRD) cancers. Other agents have also been designed to target these DDR deficiencies in cancer cells and have potential to be clinically beneficial. LP-184 is a next generation acylfulvene prodrug metabolized to an active compound by prostaglandin reductase 1 (PTGR1) that is often elevated in multiple tumors. LP-184-induced DNA lesions are likely repaired by the Nucleotide Excision Repair (NER) and Homologous Repair (HR) pathways. Reduced activities of multiple DDR-related pathways in solid tumor models correlated with increased sensitivities to LP-184 as indicated by Gene Set Enrichment Analysis. Chinese Hamster Ovary cells with NER mutations (ERCC1/2/6) were at least 4 fold more sensitive to LP-184 relative to otherwise isogenic parental cells. ERCC2 or BRCA2 depletion enhanced LP-184 sensitivity 5-8X in a metastatic prostate cancer cell line. LP-184 showed ~120 fold higher potency (IC50 77 nM) compared to Olaparib in HRD prostate cancer organoid model LuCaP 96 (BRCA2/CHEK2 inactivating mutations). Across 14 patient-derived pancreatic, lung and prostate tumor models harboring HR mutations (BRCA1/2, CHEK1/2, ATM/ATR, PALB2, PARP1/2, RAD51, FANCA/B), LP-184 showed ex vivo IC50s in the range of 30-300 nM, whereas Olaparib IC50s ranged from 1700-6900 nM. LP-184 treatment resulted in complete tumor regression (107-141% TGI) in 10/10 HR deficient triple negative breast cancer PDX models of which 7/10 were resistant Olaparib/Niraparib and to Doxorubicin/Cyclophosphamide. LP-184 treatment resulted in complete and durable tumor regression in HRD (BRCA1/ATR mutant) pancreatic cancer PDX models (112-140% TGI). In the ovarian cancer cell line OVCAR3 having HRD/NERD phenotype, LP-184 IC50 was 13 nM, Olaparib IC50 was 145 nM and their combination Bliss Synergy Score was 16 indicating synergy. LP-184 is synthetic lethal resulting from its abilities to cause unresolvable DNA damage if tumor cells express high PTGR1 and are deficient in NER/HR genes. Unlike PARPi, LP-184 has striking activity in both NERD as well as HRD cancers. These strong data have prompted the development of a soon to be launched clinical trial to translate the broad preclinical anticancer activity of LP-184 in solid tumors with HR/NER pathway defects, such as pancreatic, prostate, ovarian and breast cancers. Citation Format: Aditya Kulkarni, Jianli Zhou, Neha Biyani, Kishor Bhatia, Panna Sharma. LP-184, an acylfulvene class small molecule therapeutic, is synthetically lethal in DNA damage repair deficient cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6241.

Cytotoxic Effect of Phenylethanoid Glycosides Isolated from Plantago lanceolata L.

The aim of the study is to investigate whether the bioactive compounds isolated from P. lanceolata inflorescences, namely, phenylethanoid glucosides, acteoside, plantamajoside, and a flavonoid, isorhamnetin-3-O-rutinoside-4'-O-glucoside, possessed cytotoxic activity against the selected cancer cell lines. The potential antitumor effects of two phenylethanoid glycosides and one flavonoid were evaluated via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on seven human carcinoma cell lines (MCF-7, MDA-MB-231, Caco-2, HepG2, OVCAR-3, U138-MG, U251-MG) and one nontumorigenic mammary epithelial cell line (MCF-12A). For the first time, acteoside was studied in ovarian cancer cell line OVCAR-3, and plantamajoside in all cell lines except breast adenocarcinoma MDA-MB-281 and hepatocarcinoma HepG2. The phenylethanoid glycosides showed stronger cytotoxic activity than that of the glycoside flavonoid. Acteoside and plantamajoside, at concentrations of 200 and 300 μM, respectively, had a highly toxic effect on the selected two cancer cell lines of breast adenocarcinoma MDA-MB-231 and MCF-7, ovarian cancer cell line OVCAR-3, glioblastoma cell line U138-MG, and hepatocarcinoma cell line HepG2. Both glycosides were significantly less cytotoxic towards nontumorigenic cell line MCF-12A; the effect appeared at a concentration of 400 μM. For the first time, the activity of acteoside and plantamajoside was compared in one parallel investigation. The results are discussed against a broad background of existing knowledge on biological effects, their mechanisms, and structure-activity relationships. Phenylethanoids may be potential compounds with cytotoxic activity against the selected cancer types.

SAMHD1 expression modulates innate immune activation and correlates with ovarian cancer prognosis.

SAMHD1 is a deoxynucleotide triphosphate (dNTP) triphosphohydrolase which has been proposed as a putative prognostic factor in haematological cancers and certain solid tumours, although with controversial data. Here, we evaluate SAMHD1 function in ovarian cancer, both in vitro and in ovarian cancer patients. SAMHD1 expression was downregulated in ovarian cancer cell lines OVCAR3 and SKOV3 by RNA interference. Gene and protein expression changes in immune signalling pathways were assessed. SAMHD1 expression in ovarian cancer patients was evaluated by immunohistochemistry and survival analysis was performed according to SAMHD1 expression. SAMHD1 knockdown induced a significant upregulation of proinflammatory cytokines concomitant to increased expression of the main RNA-sensors, MDA5 and RIG-I, and interferon-stimulated genes, supporting the idea that the absence of SAMHD1 promotes innate immune activation in vitro. To assess the contribution of SAMHD1 in ovarian cancer patients, tumours were stratified in SAMHD1-low and SAMHD1-high expressing tumours, resulting in significantly shorter progression free survival (PFS) and overall survival (OS) in SAMHD1-high expression subgroup (p=0.01 and 0.04, respectively). SAMHD1 depletion correlates with increased innate immune cell signalling in ovarian cancer cells. In clinical samples, SAMHD1-low expressing tumors showed increased progression free survival and overall survival irrespective of BRCA mutation status. These results point towards SAMHD1 modulation as a new therapeutic strategy, able to enhance innate immune activation directly in tumour cells, leading to improved prognosis in ovarian cancer.

Synthesis and structural characterization of a novel palbociclib-kaempferol cocrystal with improved tabletability and synergistic antitumor activity

Palbociclib (PAL), a highly selective inhibitor of cell cycle protein-dependent kinases (CDK) 4/6, is primarily used for treating advanced breast cancer. However, the use of PAL has been limited by its poor tabletability and dose-related toxic side effects. In this study, to improve the tabletability of PAL and reduce its effective dose, a flavonoid–kaempferol (KAE) was selected as the coformer to synthesize cocrystal with PAL. The novel cocrystal PAL-KAE was prepared by antisolvent cocrystallization and characterized by various techniques including X-ray diffraction (SCXRD and PXRD), thermal analysis (DSC and TG), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), Hirshfeld surface (HS), HOMO−LUMO analysis, and molecular electrostatic potential (MEP). Powder compaction experiments indicated that PAL-KAE cocrystal significantly improved the tabletability of PAL. The cytotoxicity test suggested that PAL-KAE also exerted synergistic antitumor effect against human ovarian cancer cell line OVCAR3 in MTT assay. Docking results indicated that PAL-KAE had lower binding energy with CDK 6 than PAL. Furthermore, the cocrystal PAL-KAE exhibits excellent stability in long-term testing, accelerated testing and stressing test. This paper provides a promising strategy for improving the tabletability of PAL and enhancing its anticancer activity, thus reducing the dose-related side effects of PAL.

Synergistic effects of dendrosomal nanocurcumin and oxaliplatin on oncogenic properties of ovarian cancer cell lines by down-expression of MMPs

BackgroundContrary to the advantageous anticancer activities of curcumin (Cur), limited bioavailability and solubility hindered its efficacy. Here, nontoxic dendrosomal nano carrier with Cur was used to overcome these problems. Despite considerable antitumor properties of Oxaliplatin (Oxa), the limiting factors are drug resistance and adverse side-effects. The hypothesis of this study was to evaluate the possible synergism between dendrosomal nanocurcumin (DNC) and Oxa and these agents showed growth regulatory effects on SKOV3 and OVCAR3 cells.Methods and materialsIn the present study, colony formation, wound healing motility, cell adhesion, transwell invasion and migration assay and cell cycle arrest with or without DNC, Oxa and Combination were defined. In addition to, real time PCR and Western blot were used to analyze AKT, PI3K, PKC, JNK, P38 and MMPs mRNAs and proteins expressions. Docking of MMP-2-Cur, MMP-2-DNC and MMP-2-Oxa was performed and the results of all three complexes were simulated by molecular dynamics.ResultsOur findings illustrated that DNC had the greatest effect on cell death as compared to the Cur alone. Moreover, the growth inhibitory effects (such as cell death correlated to apoptosis) were more intense if Oxa was added followed by DNC at 4 h interval. However, insignificant effects were observed upon simultaneous addition of these two agents in both cell lines. Besides, a combination of agents synergistically alters the relative expression of MMP-9.ConclusionsThe docking results showed that His70 and Asp100 may play a key role at the MMP-2 binding site. The matrigel invasion as well as cell viability of ovarian cancer cell lines SKOV3 and OVCAR3 by DNC alone or in combination with Oxa was inhibited significantly. The inhibitory effects of these agents were due to the differential expression levels of MMP 2 and MMP 9 regulated by multiple downstream signaling cascades. From the molecular dynamic simulation studies, it was confirmed that DNC established a strong interaction with MMP-2.

Co-cultivation of human granulosa cells with ovarian cancer cells leads to a significant increase in progesterone production

PurposeIn humans, granulosa cells (GCs) are part of the follicle and nourish the growing oocyte. GCs produce estrogen and, after ovulation, progesterone. They are embedded in a multicellular tissue structure of the ovary, which consists of a variety of different cell types that are essential for the physiological function of the ovary. However, the extent to which individual ovarian cell types contribute to overall functionality has not yet been fully elucidated. In this study, we aim to investigate the effects of co-culturing human granulosa cells with ovarian cancer cells on their progesterone and estrogen production in an in vitro model.MethodsAfter seeding, the cells were stimulated with 200 µM forskolin in DMEM for 72 h and the medium of the different cell culture experiments was collected. Subsequently, progesterone and oestradiol concentrations were determined using an Elisa assay.ResultsMorphologically, it was striking that the cells self-organize and form spatially separated areas. Compared to culturing granulosa cells alone, co-culturing human granulosa cells together with the ovarian cancer cell line OvCar-3 resulted in a significant increase in progesterone production (20.3 ng/ml versus 50.2 ng/ml; p < 0.01).ConclusionsUsing a simple in vitro model, we highlight the importance of cellular crosstalk between different ovarian cells in a complex cellular network and that it strongly influences granulosa cell hormone production. This could have potential implications for the procedure of transplanting endocrine tissues after cryopreservation, as it highlights the importance of survival of all cells for the functionality of the transplanted tissue.

OR16-4 Genomic Interrogation of the Phytoestrogen Genistein in Ovarian Cancer Cells

Abstract Although studies have shown beneficial effects of the phytoestrogen genistein on reducing ovarian cancer risk, little is known at the molecular level regarding how genistein inhibits ovarian cancer growth, with estrogen receptor beta (ERb), a hormone-regulated transcription factor, receiving the bulk of the attention. When endogenous and exogenous ligands engage ERb, the receptor binds to regulatory regions in the genome known as enhancers, where it associates with coregulatory proteins, increases histone modifications associated with active enhancers, promotes chromatin looping, stimulates enhancer transcription and the production of enhancer RNAs, and ultimately regulates target gene transcription. Since signaling pathways converge at the transcriptome level, an unbiased approach is needed to examine how genistein acts to suppress ovarian cancer growth beyond ERb. We have previously used global run-on sequencing (GRO-seq), a high-throughput method that identifies the location and orientation of all transcriptionally active RNA polymerases across the genome, to identify active enhancers based on enhancer transcription and enrichment of the aforementioned genomic features. To determine the genomic responses upon genistein treatment in ovarian cancer cells, we have used PRO-seq to characterize the enhancer landscape in OVCAR3 and OVCAR4 ovarian cancer cell lines treated with genistein. The advantage of this method is the lack of a priori assumptions needed to determine which signaling pathways are regulated by genistein, since PRO-seq allows pathway determination based on the universe of active enhancers. Ultimately, this approach will allow us to uncover signaling pathways beyond ERb and determine how genistein acts as a signaling ligand to change the transcriptome in ovarian cancer cells. Presentation: Sunday, June 12, 2022 11:45 a.m. - 12:00 p.m.

Antitumor Effects of Turmeric on OVCAR-3 Ovarian Cancer Cell Lines.

Ovarian cancer is the deadliest gynecological malignancy, usually not detected until the late stages. In vitro cell culture is a method used to study the behavior of cells in a controlled environment. Turmeric has attracted the attention of scientists due to its anticancer potential. OVCAR-3 cells were cultured in RPMI medium with 100 units/mL-100 μg/mL of penicillin-streptomycin and 10% foetal bovine serum in a CO2 incubator. Turmeric extract was diluted in DMSO. Different concentrations of turmeric extract were prepared. Annexin-V staining was performed to test the translocation of phosphatidylserine to the outer side of the cell membrane as a clear indicator of apoptosis. Turmeric extract significantly reduced the viability of OVCAR-3 cells both within 24 and 48 hours of exposure. OVCAR-3 cells were treated with IC50 concentration of turmeric extract for 24 hours. 82.60% of cells were viable. The percentages of the dead, early apoptotic, and late apoptotic cells were detected to be 0.80%, 9.70%, and 6.90%, respectively. Untreated OVCAR-3 cells had migration ability. OVCAR-3 cells exposed to an IC50 concentration of turmeric extract for 24 hours did not close the scratch area. In this research, anticancer effects of turmeric have been demonstrated by different analysis methods.

IL-1β inhibitor sensitizes to olaparib in homologous recombination deficiency proficient ovarian cancer cells

Objective: To investigate the inhibitory effect of combined strategy of poly adenosine diphosphate ribose polymerase (PARP) inhibitor and interleukin-1β (IL-1β) inhibitor on homologous recombination deficiency (HRD)-proficient ovarian cancer cells. Methods: (1) HRD-proficient ovarian cancer cell lines OVCAR3 and CAOV3 were treated with PARP inhibitor olaparib. Screening by RNA sequencing analysis, the expression level of IL-1β was validated by enzyme-linked immunosorbent assay (ELISA) and western blot. (2) The dose-response curves of IL-1β inhibitor diacerein were evaluated by cell counting kit-8 (CCK-8) assays in OVCAR3 and CAOV3 cells. CCK-8 assays were further applied to determine the viabilities of OVCAR3 and CAOV3 cells. (3) To evaluate the synergistic effects of olaparib and IL-1β inhibitor in vivo, the transplanted ovarian cancer model was constructed. BALB/c-nude mice (n=16) were injected intraperitoneally with 1×107 OVACR3 cells labelled with luciferase (OVCAR3-Luc). Immunohistochemistry (IHC) assay was performed to determine nuclear antigen associated with cell proliferation (Ki-67) expression. (4) Blood routine tests, kidney and liver function tests were performed to analyze the toxic reaction of different drug treatments. The potential drug-induced injuries of vital organs including heart, liver, spleen, lungs and kidneys of nude mice were determined by hematoxylin-eosin (HE) staining. Results: (1) The RNA sequencing results showed that the mRNA level of IL-1β was the most significantly increased among the 25 differentially expressed genes in OVCAR3 cells treated with olaparib, compared to the negative control group. Olaparib treatment significantly promoted the secretion and expression of IL-1β protein in both OVACR3 and CAOV3 cells by ELISA [(36.2±3.5) and (49.5±3.5) pg/ml, respectively; all P<0.001] and western bolt (2.87±0.37 and 2.05±0.08, respectively; all P<0.01). (2) The half maximal inhibitory concentration (IC50) value of IL-1β inhibitor was determined as follows: 75 μmol/L for OVACR3 cells and 100 μmol/L for CAOV3 cells. The treatments were divided into four groups including control group, olaparib monotherapy group, IL-1β inhibitor monotherapy group and the combination therapy group. The cell viabilities of each group in OVCAR3 and CAOV3 were determined by CCK-8 assay. The data in each group were showed as follows for OVCAR3 and CAOV3 cells: (100.0±0.4)% and (100.0±3.5)% in control group; (63.1±6.2)% and (63.3±3.8)% in olaparib monotherapy group; (61.6±4.7)% and (63.8±3.5)% in IL-1β inhibitor monotherapy group; and (32.9±5.2)% and (30.0±1.3)% in the combination therapy group. The viability assay showed that the combined strategy exhibited a significant inhibition effect on OVACR3 and CAOV3 cells, compared to the monotherapy group and the control group (all P<0.01). (3) All mice with transplanted tumors of HRD-proficient ovarian cancer cells were randomly divided into four groups, and treated with four different treatments as mentioned above, respectively. After 4 weeks (on day 29), the vivo fluorescence imaging were determined. The results showed that the amount of fluorescence of transplanted tumors was mostly decreased in the combination therapy group [(0.5±0.4)×1010 p/s], compared to the control group [(4.2±1.0)×1010 p/s] or the groups treated with any single drug [(3.1±0.9)×1010, (2.2±0.9)×1010 p/s; all P<0.05]. Mice were then sacrificed under anesthesia, and all transplanted tumors detached and weighed for further investigation. The weight of transplanted tumors was significantly decreased in the combination therapy group [(0.09±0.03) g], compared to that in control group [(0.25±0.05) g] or groups treated with any single drug [(0.17±0.03), (0.19±0.04) g; all P<0.05]. The measurement of the expression of Ki-67 showed that it was significantly decreased in the combination therapy group (0.33±0.10), compared to that in the control group (1.00±0.20) or monotherapy groups (0.76±0.07, 0.77±0.12; all P<0.05). (4) There were no significant differences of body weights, blood routine test, renal and liver function tests among mice with different treatments (all P>0.05). Moreover, no significant injuries were observed in the vital organs among the four groups. Conclusions: The combination of olaparib and IL-1β inhibitor synergistically exhibits significant cytotoxicity in HRD-proficient ovarian cancer cells. Moreover, the blood routine and blood biochemistry results confirmed the biosafety of the combination of olaparib and IL-1β inhibitor.

Effect of human endogenous retrovirus-K env gene knockout on proliferation of ovarian cancer cells.

Among various human endogenous retroviruses (HERVs), the HERV-K (HML-2) group has been reported to be highly related to cancer. In pancreatic cancer cells, shRNA-mediated downregulation of HERV-K env RNA decreases cell proliferation and tumor growth through the RAS-ERK-RSK pathway; in colorectal cancer, CRISPR-Cas9 knockout (KO) of the HERV-K env gene affects tumorigenic characteristics through the nupr-1 gene. The effect of HERV-K env KO has not been studied in ovarian cancer cell lines. In this study, we analyzed the tumorigenic characteristics of ovarian cancer cell lines, including cell proliferation, migration, and invasion, and the expression patterns of related proteins after CRISPR-Cas9 KO of the HERV-K env gene. The HERV-K env gene KO was achieved using the CRISPR-Cas9 system in ovarian cancer cell lines SKOV3 and OVCAR3. Tumorigenic characteristics including cell proliferation, migration, and invasion were analyzed, and related protein expression was investigated by western blot analysis. The expression of the HERV-K env gene in KO cells was significantly reduced at RNA and protein levels, and tumorigenic characteristics including cell proliferation, migration, and invasion were significantly reduced. In HERV-K env KO SKOV3 cells, the expression of the RB protein was significantly up-regulated and the cyclin B1 protein level was significantly reduced. In contrast, in HERV-K env KO OVCAR3 cells, the level of phospho-RB protein was significantly reduced, but other protein levels were not changed. The results of this study showed that HERV-K env gene KO affects cell proliferation, invasion, and migration of ovarian cells through RB and Cyclin B1 proteins, but the specific regulation pattern can differ by cell line.

Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs)

BackgroundGenomic instability and chemoresistance can arise in cancer due to a unique form of plasticity: that of polyploid giant cancer cells (PGCCs). These cells form under the stress of chemotherapy and have higher than diploid chromosome content. PGCCs are able to then repopulate tumors through an asymmetric daughter cell budding process. PGCCs have been observed in ovarian cancer histology, including the deadly and common form high-grade serous ovarian carcinoma (HGSC). We previously discovered that drugs which disrupt the cellular recycling process of autophagy are uniquely efficacious in pre-clinical HGSC models. While autophagy induction has been associated with PGCCs, it has never been previously investigated if autophagy modulation interacts with the PGCC life cycle and this form of tumor cell plasticity.MethodsCAOV3 and OVCAR3 ovarian cancer cell lines were treated with carboplatin or docetaxel to induce PGCC formation. Microscopy was used to characterize and quantify PGCCs formed by chemotherapy. Two clinically available drugs that inhibit autophagy, hydroxychloroquine and nelfinavir, and a clinically available activator of autophagy, rapamycin, were employed to test the effect of these autophagy modulators on PGCC induction and subsequent colony formation from PGCCs. Crystal violet-stained colony formation assays were used to quantify the tumor-repopulating stage of the PGCC life cycle.ResultsAutophagy inhibitors did not prevent PGCC formation in OVCAR3 or CAOV3 cells. Rapamycin did not induce PGCC formation on its own nor did it exacerbate PGCC formation by chemotherapy. However, hydroxychloroquine prevented efficient colony formation in CAOV3 PGCCs induced by carboplatin (27% inhibition) or docetaxel (41% inhibition), as well as in OVCAR3 cells (95% and 77%, respectively). Nelfinavir similarly prevented colony formation in CAOV3 PGCCs induced by carboplatin (64% inhibition) or docetaxel (94% inhibition) as well as in OVCAR3 cells (89% and 80%, respectively). Rapamycin surprisingly also prevented PGCC colony outgrowth (52–84% inhibition).ConclusionsWhile the autophagy previously observed to correlate with PGCC formation is unlikely necessary for PGCCs to form, autophagy modulating drugs severely impair the ability of HGSC PGCCs to form colonies. Clinical trials which utilize hydroxychloroquine, nelfinavir, and/or rapamycin after chemotherapy may be of future interest.

MiR-4324 inhibits ovarian cancer progression by targeting FEN1

BackgroundOvarian cancer is one of the most lethal malignancies, with a 1.9% mortality rate worldwide. The dysregulation of the FEN1 gene and miR-4324 has been associated with cancer progression. However, the relationship between miR-4324 and-FEN1 requires further investigation.MethodsmiR-4324 and FEN1 expressions in ovarian cancer tissues and cell lines were measured via RT-qPCR. The interaction between miR-4324 and FEN1 was assessed using luciferase and RNA pull-down assays. The effects of miR-4324 and FEN1 on cell proliferation, adhesion and apoptosis were determined by CCK-8, BrdU, colony formation, cell adhesion, Caspase-3 and western blot assays in ovarian cancer cell lines CaOV3 and OVCAR3, respectively.ResultsThe results showed that miR-4324 expression was significantly decreased and FEN1 expression was enhanced in ovarian cancer tissues and cell lines. miR-4324 inhibitor promoted cell proliferation, adhesion and migration, and prevented apoptosis. Furthermore, the downregulation of FEN1 inhibited ovarian cancer cell growth and increased apoptosis. miR-4324 inhibited FEN1 expression and repressed ovarian cancer progression.ConclusionOur study found that miR-4324 inhibited FEN1 expression, suppressed cell growth, and increased apoptosis in ovarian cancer cells. Therefore, we identified miR-4324 and FEN1 as potential therapeutic targets for ovarian cancer treatment.

Wnt5A and TGFβ1 Converges through YAP1 Activity and Integrin Alpha v Up-Regulation Promoting Epithelial to Mesenchymal Transition in Ovarian Cancer Cells and Mesothelial Cell Activation.

In this paper, we investigate whether Wnt5A is associated with the TGF-β1/Smad2/3 and Hippo-YAP1/TAZ-TEAD pathways, implicated in epithelial to mesenchymal transition (EMT) in epithelial ovarian cancer. We used 3D and 2D cultures of human epithelial ovarian cancer cell lines SKOV-3, OVCAR-3, CAOV-4, and different subtypes of human serous ovarian cancer compared to normal ovary specimens. Wnt5A showed a positive correlation with TAZ and TGFβ1 in high- and low-grade serous ovarian cancer specimens compared to borderline serous and normal ovaries. Silencing Wnt5A by siRNAs significantly decreased Smad2/3 activation and YAP1 expression and nuclear shuttling in ovarian cancer (OvCa) cells. Furthermore, Wnt5A was required for TGFβ1-induced cell migration and invasion. In addition, inhibition of YAP1 transcriptional activity by Verteporfin (VP) altered OvCa cell migration and invasion through decreased Wnt5A expression and inhibition of Smad2/3 activation, which was reverted in the presence of exogenous Wnt5A. We found that the activation of TGFβ1 and YAP1 nuclear shuttling was promoted by Wnt5A-induced integrin alpha v. Lastly, Wnt5A was implicated in activating human primary omental mesothelial cells and subsequent invasion of ovarian cancer cells. Together, we propose that Wnt5A could be a critical mediator of EMT-associated pathways.

Caveolae-dependent endocytosis mediates the cellular uptake of CdTe quantum dots in ovarian cancer cell lines.

Quantum dots (QDs) are semiconductor nanocrystals that are widely used in biology due to their good optical properties. QDs, especially cadmium-based QDs, play an important role in the diagnosis and treatment of cancer due to their intrinsic fluorescence. The aim of the present study was the evaluation of the cellular uptake mechanisms of CdTe QDs in ovarian cancer cell lines. In this study, we used CdTe QDs coated with thioglycolic acid. The ovarian cancer cell lines SKOV3 and OVCAR3 were treated with different concentrations of QDs, triamterene, chlorpromazine, and nystatin, and cell viability was evaluated through the MTT test. To find the way of cellular uptake of CdTe QDs, we used the MTT test and interfering compounds in endocytic pathways. Intrinsic fluorescence and cellular internalization of CdTe QDs were assessed using flow cytometry and fluorescence microscopy imaging. The viability of CdTe QDs-treated cells dose-dependently decreased in comparison to untreated cells. To evaluate the cellular uptake pathways of CdTe QDs, in most cases, a significant difference was observed when the cells were pretreated with nystatin. The results of flow cytometry showed the cellular uptake of CdTe QDs was dose- and time-dependent. Nystatin had a measurable effect on the cellular uptake of CdTe QDs. This finding suggests that caveola-mediated endocytosis has a large portion on the internalization of CdTe QDs. According to the results of this study, CdTe QDs may have potential applications in cancer research and diagnosis.

TRIM47 promotes ovarian cancer cell proliferation, migration, and invasion by activating STAT3 signaling

ObjectivesTripartite Motif 47 (TRIM47) protein plays a prominent role in many cancers. This study aimed to investigate the biological roles of TRIM47 in ovarian cancer. MethodsTRIM47 was knocked down and overexpressed in ovarian cancer cell lines SKOV3 and OVCAR3, and the effects on proliferation, clone formation, apoptosis, invasion, and growth of xenograft tumors in nude mice were determined. The expression levels of the selected candidates were tested by western blotting and quantitative real-time PCR. ResultsTRIM47 knockdown suppressed proliferation and encourages apoptosis of ovarian cancer cells. Similarly, TRIM47 knockdown suppressed ovarian cancer cell invasion, migration, and epithelial-mesenchymal transition. Ovarian cancer cell xenograft assays demonstrated that TRIM47 knockdown significantly inhibited tumor growth. Mechanistically, TRIM47 knockdown suppressed STAT3 phosphorylation and the expression of several downstream genes, including MCL-1, MMP2, and c-MYC. Silencing of STAT3 partially prevented TRIM47–induced tumor cell proliferation and invasion. ConclusionThe present study's findings demonstrate that by activating STAT3 signaling, TRIM47 functions as an oncogene in ovarian cancer. TRIM47, therefore, appears to be a potential target for ovarian cancer prevention and/or therapy.

Effects of trans-(±)-kusunokinin on chemosensitive and chemoresistant ovarian cancer cells.

Ovarian cancer ranks eighth in cancer incidence and mortality among women worldwide. Cisplatin-based chemotherapy is commonly used for patients with ovarian cancer. However, the clinical efficacy of cisplatin is limited due to the occurrence of adverse side effects and development of cancer chemoresistance during treatment. Trans-(±)-kusunokinin has been previously reported to inhibit cell proliferation and induce cell apoptosis in various cancer cell types, including breast, colon and cholangiocarcinoma. However, the potential effects of (±)-kusunokinin on ovarian cancer remains unknown. In the present study, chemosensitive ovarian cancer cell line A2780 and chemoresistant ovarian cancer cell lines A2780cis, SKOV-3 and OVCAR-3 were treated with trans-(±)-kusunokinin to investigate its potential effects. MTT, colony formation, apoptosis and multi-caspase assays were used to determine cytotoxicity, the ability of single cells to form colonies, induction of apoptosis and multi-caspase activity, respectively. Moreover, western blot analysis was performed to determine the proteins level of topoisomerase II, cyclin D1, CDK1, Bax and p53-upregulated modulator of apoptosis (PUMA). The results demonstrated that trans-(±)-kusunokinin exhibited the strongest cytotoxicity against A2780cis cells with an IC50 value of 3.4 µM whilst also reducing the colony formation of A2780 and A2780cis cells. Trans-(±)-kusunokinin also induced the cells to undergo apoptosis and increased multi-caspase activity in A2780 and A2780cis cells. This compound significantly downregulated topoisomerase II, cyclin D1 and CDK1 expression, but upregulated Bax and PUMA expression in both A2780 and A2780cis cells. In conclusion, trans-(±)-kusunokinin suppressed ovarian cancer cells through the inhibition of colony formation, cell proliferation and the induction of apoptosis. This pure compound could be a potential targeted therapy for ovarian cancer treatment in the future. However, studies in an animal model and clinical trial need to be performed to support the efficacy and safety of this new treatment.