Ovarian Carcinoma Cell Lines Research Articles

Microtubule poleward flux as a target for modifying chromosome segregation errors

Cancer cells often display errors in chromosome segregation, some of which result from improper chromosome alignment at the spindle midplane. Chromosome alignment is facilitated by different rates of microtubule poleward flux between sister kinetochore fibers. However, the role of the poleward flux in supporting mitotic fidelity remains unknown. Here, we introduce the hypothesis that the finely tuned poleward flux safeguards against lagging chromosomes and micronuclei at mitotic exit by promoting chromosome alignment in metaphase. We used human untransformed RPE-1 cells depleted of KIF18A/kinesin-8 as a system with reduced mitotic fidelity, which we rescued by three mechanistically independent treatments, comprising low-dose taxol or codepletion of the spindle proteins HAUS8 or NuMA. The rescue of mitotic errors was due to shortening of the excessively long overlaps of antiparallel microtubules, serving as a platform for motor proteins that drive the flux, which in turn slowed down the overly fast flux and improved chromosome alignment. In contrast to the prevailing view, the rescue was not accompanied by reduction of overall microtubule growth rates. Instead, speckle microscopy revealed that the improved chromosome alignment in the rescue treatments was associated with slower growth and flux of kinetochore microtubules. In a similar manner, a low-dose taxol treatment rescued mitotic errors in a high-grade serous ovarian carcinoma cell line OVKATE. Collectively, our results highlight the potential of targeting microtubule poleward flux to modify chromosome instability and provide insight into the mechanism through which low doses of taxol rescue certain mitotic errors in cancer cells.

Three-Dimensional Model Analysis Revealed Differential Cytotoxic Effects of the NK-92 Cell Line and Primary NK Cells on Breast and Ovarian Carcinoma Cell Lines Mediated by Variations in Receptor-Ligand Interactions and Soluble Factor Profiles.

Background/objectives: The functional activity of a certain tumor determines the effectiveness of primary NK cells and NK-92 cell line-based cancer therapy; their therapeutic effectiveness against different tumors can vary. This work provides a direct simultaneous comparison of the cytotoxic effects of in vitro-activated peripheral NK (pNK) cells and NK-92 cells in spheroid models of BT-474, MCF7 and SKOV-3 carcinomas and uncovers the reasons for the differential effectiveness of NK cells against tumors. Methods: Tumor spheroids of similar size and shape, obtained from agarose molds, were incubated with NK-92 or pNK cells for 24 h. Tumor cell death was detected using flow cytometry or confocal microscopy. Cytokine production, granzyme B levels and NK cell degranulation analyses were performed, along with pNK and target-cell phenotypic characterization. Results: While NK-92 and pNK cells lysed BT-474 spheroids with comparably low efficiency, pNK cells were more capable of eliminating MCF7 and SKOV-3 spheroids than NK-92 cells were. The results of the functional and phenotypic analyses strongly support the participation of the NKG2D-NKG2DL pathway in pNK cell activation induced by the most sensitive cytotoxic attack on SKOV-3 spheroids, whereas the CX3CR1-CX3CL1 axis appears to be involved in the pNK reaction against MCF-7 spheroids. Conclusions: We provide a new approach for the preliminary identification of the most promising NK cell receptors that can alter the effectiveness of cancer therapy depending on the specific tumor type. Using this approach, NK-92 cells or pNK subsets can be selected for further accumulation and/or genetic modification to improve specificity and reactivity.

B-308 Analytical and clinical performance of a novel ovarian cancer screening test in asymptomatic women

Abstract Background Ovarian cancer (OC) ranks among the deadliest gynecological cancers, often diagnosed at advanced stages when treatment options are limited. Costly sequencing methods and established immunoassay and imaging-based methods lack early-stage sensitivity, highlighting the need for a cost-effective test with improved performance. Extracellular vesicles (EVs) produced in abundance by all cells can potentially improve test sensitivity and detection of multiple colocalized cancer specific markers can improve test specificity. The randomized controlled trial UKCTOCS (United Kingdom Collaborative Trial of Ovarian Cancer Screening) where over 200,000 postmenopausal asymptomatic women were randomly assigned to three arms revealed no significant reduction in mortality with OC screening. We report on an EV-based OC Test performance in serum samples from asymptomatic postmenopausal women in UKCTOCS. Methods The OC Test was previously locked (biomarkers, method, cutoff) and validated in an independent cohort. We evaluated the OC Test in a blinded case-control study nested within the no screening (NS) and annual ultrasound screening (USS) arms of UKCTOCS. All UKCTOCS participants provided a blood sample at study entry. Cases were all women who developed high grade serous ovarian cancer (HGSOC) within 36 months following sample donation (spun within 28 hours). They were matched by age at collection (+/-2 years) to women who had no OC detected either before or within the trial follow up period (controls). Where a case had >1 sample within the 0-36 months period, the control sample was matched to the case sample closest to diagnosis. The OC Test was run with a locked method and classifier algorithm utilizing 3 independent biomarker combinations tested in duplicate. OC Test results were calculated based on a previously determined cutoff prior to unblinding of clinical outcomes. Run controls were prepared by harvesting conditioned media from COV413A cell cultures (an ovarian epithelial-serous carcinoma cell line), purifying EVs and spiking into pre-screened OC Test negative human plasma at three levels. Run controls were run twice per day, one replicate per run for 17 days. Results We retrospectively assessed 116 samples from 108 cases (63 clinical Dx NS, 15 clinical Dx USS, 30 screen detected USS) women with HGSOC and 1310 samples from 1310 controls. OC test sensitivity for HGSOC was 84% (36/43; 95% CI 69%-93%), Stage I/II sensitivity was 89% (8/9; 95% CI 51%-99%) and the AUC (area under the curve) was 0.93 for case samples drawn 0-12 months prior to clinical Dx and all control samples. The OC Test specificity was 98% (1099/1176, 95% CI 97%-98%). Within laboratory CVs were 20.9%, 12.7% and 0.6% for low negative, high negative and high positive run controls respectively. Data from 143 samples (10%, 9 cases, 134 controls) were excluded due to operator error, low sample volume or quality issues. Conclusions The OC Test is capable of highly sensitive detection of HGSOC within one year of clinical diagnosis including at the earliest stages of disease while maintaining exceptionally high specificity. The results demonstrate the potential application of the OC Test for ovarian cancer screening in asymptomatic women.

The first-in-class pro-apoptotic peptide PEP-010 is effective in monotherapy and in combination with paclitaxel on resistant ovarian adenocarcinoma cell models.

Ovarian adenocarcinoma is the gynecological malignancy with the worst prognosis and the highest mortality rate. In the first stages of treatment, chemotherapy results effective, but its prolonged use and high doses lead to the appearance of resistance to treatments and relapse in most patients, representing a major challenge for clinicians. We developed PEP-010, a cell penetrating proapoptotic peptide disrupting the protein-protein interaction between caspase-9 and protein phosphatase 2A, thereby leading to the recovery of their activity in the apoptotic pathway. MTT assay or Annexin-V/Propidium Iodide staining and flow cytometry analysis were used to assess sensitivity to chemotherapies and apoptosis after treatment with PEP-010 in monotherapy or in combination with paclitaxel in ovarian carcinoma cell lines. DNA damage was assessed by immunofluorescence using γH2AX marker. We show here that PEP-010 effectively induces cell death in monotherapy on in up to 55% of cells from ovarian adenocarcinoma cell models resistant to different chemotherapies. Moreover, when used in combination with paclitaxel, one of the therapeutic options for recurrent ovarian carcinoma, PEP-010 showed a beneficial effect leading to the reduction of the IC50 of paclitaxel of 2.2 times and to apoptosis in 87% of cells. The described results suggest the potential therapeutic interest for PEP-010 and lead to the choice of ovarian adenocarcinoma as one of the major indications of the ongoing clinical trial.

Cell-intrinsic platinum response and associated genetic and gene expression signatures in ovarian cancer cell lines and isogenic models.

Ovarian cancers are still largely treated with platinum-based chemotherapy as the standard of care, yet few biomarkers of clinical response have had an impact on clinical decision making as of yet. Two particular challenges faced in mechanistically deciphering platinum responsiveness in ovarian cancer have been the suitability of cell line models for ovarian cancer subtypes and the availability of information on comparatively how sensitive ovarian cancer cell lines are to platinum. We performed one of the most comprehensive profiles to date on 36 ovarian cancer cell lines across over seven subtypes and integrated drug response and multiomic data to improve on our understanding of the best cell line models for platinum responsiveness in ovarian cancer. RNA-seq analysis of the 36 cell lines in a single batch experiment largely conforms with the currently accepted subtyping of ovarian cancers, further supporting other studies that have reclassified cell lines and demonstrate that commonly used cell lines are poor models of high-grade serous ovarian carcinoma. We performed drug dose response assays in the 32 of these cell lines for cisplatin and carboplatin, providing a quantitative database of IC 50 s for these drugs. Our results demonstrate that cell lines largely fall either well above or below the equivalent dose of the clinical maximally achievable dose (C max ) of each compound, allowing designation of cell lines as sensitive or resistant. We performed differential expression analysis for high-grade serous ovarian carcinoma cell lines to identify gene expression correlating with platinum-response. Further, we generated two platinum-resistant derivatives each for OVCAR3 and OVCAR4, as well as leveraged clinically-resistant PEO1/PEO4/PEO6 and PEA1/PEA2 isogenic models to perform differential expression analysis for seven total isogenic pairs of platinum resistant cell lines. While gene expression changes overall were heterogeneous and vast, common themes were innate immunity/STAT activation, epithelial to mesenchymal transition and stemness, and platinum influx/efflux regulators. In addition to gene expression analyses, we performed copy number signature analysis and orthogonal measures of homologous recombination deficiency (HRD) scar scores and copy number burden, which is the first report to our knowledge applying field-standard copy number signatures to ovarian cancer cell lines. We also examined markers and functional readouts of stemness that revealed that cell lines are poor models for examination of stemness contributions to platinum resistance, likely pointing to the fact that this is a transient state. Overall this study serves as a resource to determine the best cell lines to utilize for ovarian cancer research on certain subtypes and platinum response studies, as well as sparks new hypotheses for future study in ovarian cancer.

Phytochemical Composition, In Vitro Antioxidant, and Anticancer Activities of Cold Pressed Oil from Sesame (Sesamum indicum L.) from the Kingdom of Saudi Arabia

Sesame seed oil (SSO) has long been used in many complementary and alternative medicine systems to treat a variety of maladies and ailments. SSO was obtained by cold pressing Sesamum indicum L. (sesame) seeds. Sesamum indicum L. (sesame) oil’s chemical constituents identified with GC-MS. Using the DDPH and MTT assays, respectively, the antioxidant and anticancer activities were investigated. phytosterols, (β-sitosterol, Campesterol, and Stigmasterol), lignans such as sesamin, fatty acids such as Oleic Acid, Esters of fatty acid, such as Hexadecanoic acid, methyl ester, Linoleic acid ethyl ester, flavonoids such as 3′,4′,7Trimethylquercetin, 6,8-di-c-a-glucosylluteolin, the carboxylic ester glycidyl oleate, alcoholic compounds, 1-heptatriacotanol, were the active chemical compounds detected in Sesamum indicum L. (sesame) oil. SSO has a high tocopherol profile. SSO showed antioxidant properties with DDPH radical scavenging of 63.1% relative to standard beta hydroxyl butyrate (BHT). The anticancer effects of SSO against Ovarian carcinoma (A-2780) cell line with IC50 = 1.68 ± 0.3 μg/mL. Sesamum indicum L. (sesame) oil’s chemical constituents identified with GC-MS showed antioxidant and anticancer properties.

Liposome-encapsulated progesterone efficiently suppresses B-lineage cell proliferation

Progesterone suppresses several ancient pathways in a concentration-dependent manner. Based on these characteristics, progesterone is considered a candidate anticancer drug. However, the concentration of progesterone used for therapy should be higher than the physiological concentration, which makes it difficult to develop progesterone-based anticancer drugs. We previously developed liposome-encapsulated progesterone (Lipo-P4) with enhanced anticancer effects, which strongly suppressed triple-negative breast cancer cell proliferation in humanized mice. In this study, we aimed to clarify whether Lipo-P4 effectively suppresses the proliferation of B-lineage cancer cells. We selected six B-cell lymphoma and two myeloma cell lines, and analyzed their surface markers using flow cytometry. Next, we prepared liposome-encapsulated progesterone and examined its effect on cell proliferation in these B-lineage cancer cells, three ovarian clear cell carcinoma cell lines, two prostate carcinoma cell lines, and one triple-negative breast cancer adenocarcinoma cell line. Lipo-P4 suppressed the proliferation of all cancer cell lines. All B-lineage cell lines, except for the HT line, were more susceptible than the other cell types, regardless of the expression of differentiation markers. Empty liposomes did not suppress cell proliferation. These results suggest that progesterone encapsulated in liposomes efficiently inhibits the proliferation of B-lineage cells and may become an anticancer drug candidate for B-lineage cancers.

Establishment of a human ovarian clear cell carcinoma cell line mutant in PIK3CB but not PIK3CA

A human ovarian clear cell carcinoma cell line was established from a 46-year-old Japanese woman. That line, designated MTC-22, has proliferated continuously for over 6 months in conventional RPMI 1640 medium supplemented with 10% foetal bovine serum and has been passaged over 50 times. MTC-22 doubling-time is ~ 18 h, which is much shorter than most ovarian clear cell carcinoma lines reported to date. Morphologically, MTC-22 cells exhibit polygonal shapes and proliferate to form a monolayer in a jigsaw puzzle-like arrangement without contact inhibition. Ultrastructurally, cells exhibit numerous intracytoplasmic glycogen granules and well-developed mitochondria. G-band karyotype analysis indicated that cells have a complex karyotype close to tetraploid. We observed that the expression pattern of a series of ovarian carcinoma-related molecules in MTC-22 cells was identical to that seen in the patient’s tumour tissue. Notably, MTC-22 cells, and the patient’s carcinoma tissue, expressed low-sulphated keratan sulphate recognised by R-10G and 294-1B1 monoclonal antibodies, a hallmark of non-mucinous ovarian carcinoma, and particularly of clear cell ovarian carcinoma. Moreover, characteristic point mutations—one in ARID1A, which encodes the AT-rich interaction domain containing protein 1A, and the other in PIK3CB, which encodes the catalytic subunit of phosphoinositide 3-kinase—were seen in the patient’s tumour tissue and retained in MTC-22 cells. Collectively, these findings indicate that MTC-22 cells could serve as a valuable tool for investigating the pathophysiology of ovarian clear cell carcinoma, particularly that harbouring PIK3CB mutations, and for developing and validating new diagnostic and therapeutic approaches to this life-threatening malignancy.

Structural Elucidation and Prognostic Relevance of 297-11A-Sulfated Glycans in Ovarian Carcinoma

Ovarian carcinoma is usually diagnosed at an advanced stage with peritoneal dissemination and/or lymph node metastasis, and the prognosis for such advanced carcinoma is very poor. Therefore, new biomarkers to predict patient prognosis are needed. Miyamoto et al. previously showed that keratan sulfate (KS) detected by the 5D4 monoclonal antibody was expressed in ovarian carcinoma. However, the detailed structure of such KS was not determined, and the biological significance of this finding remained to be clarified. We previously generated the 297-11A monoclonal antibody, which recognizes galactose (Gal)-6-O-sulfated N-acetyllactosamine (LacNAc) located at the nonreducing terminus. Because the 297-11A epitope overlaps with that of 5D4, here we chose to use the 297-11A antibody as a tool to analyze KS and related structures. We conducted immunohistochemical analysis of 98 ovarian carcinoma cases with 297-11A antibody combined with a series of glycosidases and performed mass spectrometry analysis of the human serous ovarian carcinoma cell line OVCAR-3 to deduce the glycan structure of 297-11A-sulfated glycans. We also performed western blot analysis to assess a potential association of 297-11A-sulfated glycans with the mucin core protein mucin 16 (MUC16; also known as cancer antigen 125 (CA125)). Finally, we examined the relationship between 297-11A expression and patient prognosis. Consequently, 297-11A-sulfated glycans were primarily expressed in serous and endometrioid carcinomas and poorly expressed in mucinous and clear cell carcinomas. We reveal that structurally, 297-11A-sulfated glycans expressed in ovarian carcinoma are O-glycans carrying partially sialylated, Gal-6-O-sulfated LacNAc and that these glycans are likely displayed on MUC16 mucin core proteins. Of clinical importance is that expression of 297-11A-sulfated glycans correlated with shorter progression-free survival in patients. Thus, 297-11A-sulfated glycans may serve as a predictor of ovarian carcinoma recurrence.

Integrated analysis of scRNA-seq and bulk RNA-seq identifies FBXO2 as a candidate biomarker associated with chemoresistance in HGSOC

BackgroundHigh-grade serous ovarian carcinoma (HGSOC) is the most prevalent and aggressive histological subtype of epithelial ovarian cancer. Around 80% of individuals will experience a recurrence within five years because of resistance to chemotherapy, despite initially responding well to platinum-based treatment. Biomarkers associated with chemoresistance are desperately needed in clinical practice. MethodsWe jointly analyzed the transcriptomic profiles of single-cell and bulk datasets of HGSOC to identify cell types associated with chemoresistance. Copy number variation (CNV) inference was performed to identify malignant cells. We subsequently analyzed the expression of candidate biomarkers and their relationship with patients' prognosis. The enrichment analysis and potential biological function of candidate biomarkers were explored. Then, we validated the candidate biomarker using in vitro experiments. ResultsWe identified 8871 malignant epithelial cells in a single-cell RNA sequencing dataset, of which 861 cells were associated with chemoresistance. Among these malignant epithelial cells, FBXO2 (F-box protein 2) is highly expressed in cells related to chemoresistance. Moreover, FBXO2 expression was found to be higher in epithelial cells from chemoresistance samples compared to those from chemosensitivity samples in a separate single-cell RNA sequencing dataset. Patients exhibiting elevated levels of FBXO2 experienced poorer outcomes in terms of both overall survival (OS) and progression-free survival (PFS). FBXO2 could impact chemoresistance by influencing the PI3K-Akt signaling pathway, focal adhesion, and ECM-receptor interactions and regulating tumorigenesis. The 50% maximum inhibitory concentration (IC50) of cisplatin decreased in A2780 and SKOV3 ovarian carcinoma cell lines with silenced FBXO2 during an in vitro experiment. ConclusionsWe determined that FBXO2 is a potential biomarker linked to chemoresistance in HGSOC by combining single-cell RNA-seq and bulk RNA-seq dataset. Our results suggest that FBXO2 could serve as a valuable prognostic marker and potential target for drug development in HGSOC.

Abstract A038: Epigenetic compound library screen of ovarian clear cell carcinoma cell line models identifies decreased cell viability following treatment with the Bruton tyrosine kinase inhibitor ibrutinib

Abstract Ovarian clear cell carcinoma (OCCC) is a difficult to treat histological subtype of ovarian cancer, demonstrating resistance to standard chemotherapy. New approaches are required in order to identify compounds with the potential to target this tumor. OCCC are characterized by mutations in ARID1A and/or ARID1B (AT-rich interactive domain-containing proteins) that encode for members of the mammalian SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex. We have used an epigenetic library consisting of 160 compounds that targets epigenetic readers, writers, erasers, and transcriptional modulators, to screen seven OCCC cell line models (RMG1, JHOC5, OVTOKO, OVMANA, OVISE, OV207 and TOV21G) and three non-OCCC ovarian cancer cell line models (A2780, COV434 and OVCAR8). Cell lines were seeded in triplicate into 96-well plates and treated with all 160 compounds at both 5 µM and 0.5 µM, as well as with a DMSO vehicle control, for 72 hours. Following treatment, cell viability was determined using a MTS assay and each data point normalized to the DMSO control. Based on the results of viability assays, drugs were sought that preferentially targeted OCCC relative to non-OCCC cell lines. Drugs that increased cell proliferation in two or more of the OCCC lines were removed from the analysis, as were drugs that did not impact upon viability. Drug 152, ibrutinib, inhibited cell viability between 53 to 78% in 6 of the 7 OCCC cell lines at 5 µM, with an average inhibition of 64%. In non-OCCC cell lines, the average cell viability after treatment with 5 µM ibrutinib was 91% (range, 84 – 103%). Ibrutinib is a small molecule inhibitor of Bruton tyrosine kinase (BTK) that has been approved by the FDA for treatment of a number of hematological malignancies including mantle cell lymphoma, small lymphocytic lymphoma and chronic lymphocytic leukemia. BTK functions in B-cell receptor signaling, regulating B cell proliferation and survival. Ibrutinib works by irreversibly blocking the kinase activity of BTK. Interestingly, ibrutinib binding sites are also present in other tyrosine kinases, including EGFR, raising the possibility of off-target effects that can function to decrease tumor cell viability. In addition to hematological malignancies, ibrutinib is being investigated in clinical trials for the potential to impact on solid tumors including non-small cell lung cancer, certain oesophagogastric carcinoma, advanced carcinoid and pancreatic neuroendocrine tumors, squamous cell carcinoma of the head and neck, renal cell carcinoma and glioblastoma, including in some cases in combination with other therapeutic drugs including cisplatin, temozolomide and nivolumab. Following organoid drug testing, off-label use of ibrutinib in a patient with low-grade serous ovarian cancer has recently shown promising results. Research is on-going to determine the effectiveness of ibrutinib in pre-clinical models of OCCC, including analyses of the effectiveness of ibrutinib in ARID1A and ARID1B wild-type and mutant CRISPR-engineered isogenic cell line panels of OCCC. Citation Format: Yue Ma, Kristie Ann Dickson, Natisha Field, Tao Xie, Nham Tran, Deborah Joy Marsh. Epigenetic compound library screen of ovarian clear cell carcinoma cell line models identifies decreased cell viability following treatment with the Bruton tyrosine kinase inhibitor ibrutinib [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A038.

Abstract B058: Targeting mediator kinase CDK8/19 potentiates chemotherapeutic responses, reverses tumor growth, and prolongs survival from ovarian clear cell carcinoma

Abstract Ovarian clear cell carcinomas (OCCC) are a rare histological subtype of epithelial ovarian cancer with known treatment resistance, a feature associated with transcriptional reprogramming. CDK8 and CDK19 Mediator Kinases, which are isoforms of the Mediator Kinase – enzymatic components of a regulatory module that binds to the transcriptional Mediator complex –and not a part of the overall transcription machinery, have been previously reported to modify oncogenic transcription factors. Inhibition of the kinase activity of CDK8/19 was shown to prevent and reverse chemoresistance in other cancers with two CDK8/19 inhibitors currently in clinical trials for non–ovarian cancers. Based on the unmet need for OCCCs, we assessed CDK8/19 expression in a panel of OCCC primary and metastatic tissues and utilized cell line models for investigation into the therapeutic potential of CDK8/19 inhibitors in preventing tumorigenesis and synergizing with existing chemotherapeutics. The synergistic effects of SNX631-6, a preclinical CDK8/19 inhibitor drug candidate, were tested in vitro using three clear cell (ES2, TOV-21-G, RMG1) and one non-clear cell (SKOV3) ovarian carcinoma cell lines. A sub-inhibitory concentration 50 (IC50) concentration of SNX631-6 (500 nM) strongly potentiated effects of cisplatin, carboplatin, and paclitaxel. Additionally, cells demonstrating platinum resistance reverted to platinum sensitivity when exposed to the same concentration of SNX631-6. In vivo antitumor effects and tolerability of oral SNX631-6 were assessed in Foxn1nu homozygous female mice injected subcutaneously with ES2+luc cells. Mice were treated with a vehicle (n=10), intraperitoneal (IP) cisplatin (2 mg/kg) twice weekly (n=9), SNX631-6 medicated diet (330 ppm) orally ad libitum (n=10), or IP cisplatin combined with SNX631 diet (n=10) until tumors reached a predetermined critical volume of 2500mm3. In ES2+luc injected mice, significant tumor growth inhibition relative to vehicle occurred in mice treated with a combination of IP cisplatin and SNX631-6 (p<0.001) or SNX631-6 alone (p<0.01). Additionally, the use of IP cisplatin and SNX631-6 significantly delayed time-to-critical-volume compared to vehicle (p=0.0001). SNX631-6 alone and in combination with IP cisplatin was well tolerated in all mice based on body weight measurements. In summary, SNX631-6 potentiated platinum responses in vitro and demonstrated strong in vivo antitumor effects, including tumor regression, when used as monotherapy and in combination with platinum-based therapy. These data suggest that therapeutically achievable doses of SNX631-6 may provide clinical benefit, prevent tumorigenesis, and reverse platinum resistance in patients with OCCC. Citation Format: Wade Barton, Asha Kumari, Gary School, Zachary Mack, Liz Macias Quintero, Karthik Rangavajhula, Rebecca Arend, Mengqian Chen, Eugenia V. Broude, Karthikeyan Mythreye. Targeting mediator kinase CDK8/19 potentiates chemotherapeutic responses, reverses tumor growth, and prolongs survival from ovarian clear cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B058.

Novel (±)-trans-β-lactam ureas: Synthesis, in silico and in vitro biological profiling.

A diastereomeric mixture of racemic 3-phthalimido-b-lactam 2a/2b was synthesized by the Staudinger reaction of carboxylic acid activated with 2-chloro-1-methylpyridinium iodide and imine 1. The amino group at the C3 position of the b-lactam ring was used for further structural upgrade. trans-b-lactam ureas 4a-t were prepared by the condensation reaction of the amino group of b-lactam ring with various aromatic and aliphatic isocyanates. Antimicrobial activity of compounds 4a-t was evaluated in vitro and neither antibacterial nor antifungal activity were observed. Several of the newly synthesized trans-b-lactam ureas 4a-c, 4f, 4h, 4n, 4o, 4p, and 4s were evaluated for in vitro antiproliferative activity against liver hepatocellular carcinoma (HepG2), ovarian carcinoma (A2780), breast adenocarcinoma (MCF7) and untransformed human fibroblasts (HFF1). The b-lactam urea 4o showed the most potent antiproliferative activity against the ovarian carcinoma (A2780) cell line. Compounds 4o and 4p exhibited strong cytotoxic effects against human non-tumor cell line HFF1. The b-lactam ureas 4a-t were estimated to be soluble and membrane permeable, moderately lipophilic molecules (logP 4.6) with a predisposition to be CYP3A4 and P-glycoprotein substrates. The tools PASS and SwissTargetPrediction could not predict biological targets for compounds 4a-t with high probability, pointing to the novelty of their structure. Considering low toxicity risk, molecules 4a and 4f can be selected as the most promising candidates for further structure modifications.

CLCN3 in mediating the proliferation of human ovarian cancer cells.

Chloride channel-3 (CLCN3), a crucial component of the voltage-gated chloride channel family, is implicated in numerous physiological and pathophysiological processes. This study aimed to investigate the characteristics of CLCN3 in pancancer and its influence on the immune response through the use of a range of databases. Concurrently, we assessed the impact of CLCN3 on the proliferation of ovarian cancer (OC) cells and explored its potential mechanisms. We employed the Tumor Immune Estimation Resource (TIMER) 2.0 and Clinical Proteomic Tumor Analysis Consortium (CPTAC) databases to examine the messenger RNA (mRNA) and the protein expression of CLCN3 across various cancers. The prognostic significance of CLCN3 was evaluated using the Gene Expression Profiling Interactive Analysis 2.0 (GEPIA 2.0) database. The University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) facilitated the analysis of CLCN3 promoter methylation levels. The association between CLCN3 expression and tumor-infiltrating immune cells was investigated using various algorithms. The cBioportal database facilitated the analysis of CLCN3 mutations and mutation sites across various cancers. The Tumor-Immune System Interactions Database (TISIDB) database was employed to explore the correlation between CLCN3 expression and immune or molecular subtypes across a variety of cancer types. We collected ovarian tissue samples, encompassing both normal ovarian and OC tissues. The human OC cell lines, SKOV3 cells and OVCAR433 cells, were cultured. CLCN3 expression was determined via reverse-transcription quantitative polymerase chain reaction (RT-qPCR), while phosphatidylinositol 3-kinase/Akt kinase (PI3K/AKT) expression was detected using Western blot. We utilized small interfering RNA (siRNA) technology to suppress CLCN3 expression. The proliferative capacity of SKOV3 and OVCAR433 cells was assessed using the Cell Counting Kit 8 (CCK-8) assay. CLCN3 demonstrated an aberrant expression in a number of cancer types and was markedly reduced in OC tissues. Poor prognosis in cervical squamous cell cancer and myeloid leukemia was linked to excessive expression of CLCN3. The examination of immune cell infiltration, which included CD8+ T cells, B cells, T regulatory cells, and cancer-associated fibroblast cells, showed a strong association with aberrant CLCN3 expression. Following the use of siRNA technology, the ability of the ovarian carcinoma cell line SKOV3 and OVCAR433 to proliferate as well as the expression of PI3K/AKT both increased. CLCN3 is a possible biomarker for immune-related processes and the prognosis of cancer, and the PI3K/AKT signaling pathway may affect OC cells' ability to proliferate.

Novel Bidentate Amine Ligand and the Interplay between Pd(II) and Pt(II) Coordination and Biological Activity.

Pt(II) and Pd(II) coordinating N-donor ligands have been extensively studied as anticancer agents after the success of cisplatin. In this work, a novel bidentate N-donor ligand, the N-[[4-(phenylmethoxy)phenyl]methyl]-2-pyridinemethanamine, was designed to explore the antiparasitic, antiviral and antitumor activity of its Pt(II) and Pd(II) complexes. Chemical and spectroscopic characterization confirm the formation of [MLCl2 ] complexes, where M=Pt(II) and Pd(II). Single crystal X-ray diffraction confirmed a square-planar geometry for the Pd(II) complex. Spectroscopic characterization of the Pt(II) complex suggests a similar structure. 1 H NMR, 195 Pt NMR and HR-ESI-MS(+) analysis of DMSO solution of complexes indicated that both compounds exchange the chloride trans to the pyridine for a solvent molecule with different reaction rates. The ligand and the two complexes were tested for in vitro antitumoral, antileishmanial, and antiviral activity. The Pt(II) complex resulted in a GI50 of 10.5 μM against the NCI/ADR-RES (multidrug-resistant ovarian carcinoma) cell line. The ligand and the Pd(II) complex showed good anti-SARS-CoV-2 activity with around 65 % reduction in viral replication at a concentration of 50 μM.

Spheroid architecture strongly enhances miR-221/222 expression and promotes oxidative phosphorylation in an ovarian cancer cell line through a mechanism that includes restriction of miR-9 expression.

Tumor cell spheroids are organized multicellular structures that form during the expansive growth of carcinoma cells. Spheroids formation is thought to contribute to metastasis by supporting growth and survival of mobile tumor cell populations. We investigated how spheroid architecture affects OXPHOS activity, microRNA expression, and intraperitoneal survival of an ovarian carcinoma cell line using high resolution respirometry, quantitative RT-PCR, and a rodent intraperitoneal growth model. Rates of oxidative phosphorylation/respiration per cell of cells growing as spheroids were nearly double those of a variant of the same cell type growing in suspension as loosely aggregated cells. Further, inhibition of spheroid formation by treatment with CDH2 (N-cadherin) siRNA reduced the rate of OXPHOS to that of the non-spheroid forming variant. Cells growing as spheroids showed greatly enhanced expression of miR-221/222, an oncomiR that targets multiple tumor suppressor genes and promotes invasion, and reduced expression of miR-9, which targets mitochondrial tRNA-modification enzymes and inhibits OXPHOS. Consistent with greater efficiency of ATP generation, tumor cells growing as spheroids injected into the nutrient-poor murine peritoneum survived longer than cells growing in suspension as loosely associated aggregates. The data indicate that growth in spheroid form enhances the OXPHOS activity of constituent tumor cells. In addition, spheroid architecture affects expression of microRNA genes involved in growth control and mitochondrial function. During the mobile phase of metastasis, when ovarian tumor cells disperse through nutrient-poor environments such as the peritoneum, enhanced OXPHOS activity afforded by spheroid architecture would enhance survival and metastatic potential.

Silencing HDAC1 Expression Regulates Extracellular Regulated Protein Kinases/Mitogen-Activated Protein Kinase Signaling Activation and Enhances Chemosensitivity of Ovarian Carcinoma

Objective: Cisplatin (DDP)-based therapy is frequently employed as a first-line chemotherapy for ovarian carcinoma (OC). ERK/mitogen-activated protein kinase (MAPK), as a widely studied axis with cancer-promoting action, is reported to be essential in mediating chemoresistance in many cancer cells. This study investigated the effect of HDAC1 expression on the chemosensitivity of OC via regulation of the ERK/MAPK axis. Methods: Fifty-eight cases of OC and adjacent tissue specimens were acquired, as well as the survival data of patients with OC in the Cancer Genome Atlas. To detect HDAC1 in tissues, a HDAC1-DNA probe was constructed, and patients with different HDAC1 levels were analyzed for survival. HDAC1 levels were also detected in SKOV3 and SKOV3/DDP cells. HDAC1 siRNA was used to interfere with HDAC1 expression, after which the sensitivity to DDP sensitivity and changes in apoptosis of tumor cells were detected using the MTT assay and flow cytometry, respectively. Western blotting quantified the expression of ERK/MAPK axis-related proteins. Results: HDAC1 was highly expressed in OC tissues, with a high expression significantly related to adverse outcomes of patients. Compared with that in SKOV3 cells, SKOV3/DDP cells showed decreased sensitivity to DDP and increased HDAC1 expression. After transfection of HDAC1-siRNA, SKOV3/DDP cell sensitivity to DDP increased, p38 MAPK, ERK1/2, and JNK phosphorylation levels decreased, and the apoptosis rate increased markedly; transfection with an ERK agonist reversed the inhibitory action of HDAC1-siRNA against cells. Conclusions: Silencing HDAC1 expression significantly reduced the chemoresistance of DDP-resistant OC cell lines and promoted apoptosis, and its mechanism is related to inhibition of ERK/MAPK activation.

A PILOT IN VITRO STUDY ON THE EFFECT OF BLUE HONEYSUCKLE EXTRACT ON HUMAN NON-CANCEROUS AND CANCEROUS OVARIAN CELLS

Honeysuckle (Lonicera caerulea L.) is utilized in Chinese, Japanese, and Russian folk medicines for centuries. The berries referred to as blue honeysuckle are recently gaining popularity in central and southern Europe due to remarkable therapeutic potential and widespread use as an ingredient in dietary supplements. The primary objective of this investigation was to examine the impact of blue honeysuckle extract on human ovarian cells – both non-cancerous HGL5 (immortalized human ovarian granulosa cell line) and cancerous OVCAR-3 (human ovarian carcinoma cell line). Cell viability, secretion of steroid hormones (17ß-estradiol and progesterone), human transforming growth factor-β2 (TGFβ-2) and its receptor (TGFBR2), and the level of apoptosis-inducing factor (AIF) were examined in ovarian granulosa cells HGL5, and the same parameters (excepting the steroid hormones) were assessed in ovarian epithelial cancer cells OVCAR-3. Blue honeysuckle ethanolic extract was applied to cells for 24 hours. AlamarBlueTM assay was used to assess cell viability, whereas secretion of steroid hormones was evaluated by ELISA. ELISA was also used to assess the levels of TGF-β2, its receptor TGFBR2 and AIF in HGL5 and OVCAR-3 cell lysates. Based on a range of doses ranging from 5 – 100 μg/mL, the extract from blue honeysuckle had no adverse effects on the viability of human ovarian cells. Futuremore, it did not affect their secretory activity – be it the release of steroid hormones or that of growth factor TGF-β2 and its receptor TGFBR2, nor did induce apoptosis (as revealed by the AIF). The present pilot study for the first time reports the effect of blue honeysuckle extract on ovarian cell models using both non-cancerous cells HGL5 and cancerous OVCAR-3 cells. These findings provide initial insights into the safety and potential utility of blue honeysuckle extract, warranting further investigation into its therapeutic applications. In further research, it is possible to examine a longer exposure time to see if it affects the cells.

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Abstract IA014: From targeted phenotypic screen to NXP800: A clinical stage activator of the integrated stress response for the treatment of ARID1A-mutated ovarian carcinoma

Mice ovarian microbiome investigation divulges prospective fount of anticancer and antimicrobial metabolites

The impact of bacterial microbiome metabolites on human health, most particularly on the prevention and treatment of cancer, has gained significant interest in recent times. The study focused on the isolation of bacterial flora associated with the ovaries of 8-week-old mice and examined its ability to produce metabolites with potential antibacterial, antioxidant, and anticancer properties. The isolates were identified based on biochemical and 16S rRNA sequencing. The ethyl acetate extract from isolates was screened for antimicrobial and antioxidant capacity. The 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of the selected isolate against the ovarian carcinoma cell lines SKOV-3 and PA-1. The ethyl acetate extract from Bacillus velezensis OM03 exhibited significant antibacterial activity against all the tested bacteria, with a MIC value ranging from 50 to 100 µg/mL. Furthermore, the extracts demonstrated hydrogen peroxide and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities at rates of 88.50% and 87.78%, respectively. The extracts displayed substantial concentration-dependent antiproliferative/cytotoxic activities against SKOV-3 and PA-1 cell lines after 24 and 48 h of treatments, respectively. Further chemical analysis of the extract using HR-LCMS revealed the presence of bioactive compounds such as myriocin, 2,3-diethylpyrazine, dihydrodeoxystreptomycin, cyclo (L-Phe-L-Pro), C16 sphinganine, and other twenty-nine compounds that have been formerly reported and are accountable for the targeted activities. Bacillus velezensis OM03 may be further investigated for the creation of novel therapeutics, particularly for the treatment of ovarian cancer.