TOV-21G Cells Research Articles

TNFRSF10D expression as a potential biomarker for cisplatin-induced damage and ovarian tumor relapse prediction

Among gynecological malignancies, ovarian cancer (OC) presents the most challenging diagnostic scenario. Despite exhaustive efforts, up to 90% of patients treated with taxane/platinum-based chemotherapy experience relapse, leading to poor survival rates. Identifying new molecular markers that can characterize disease aggressiveness, chemoresistance, recurrence risk, and metastasis is crucial. This study aimed to assess the susceptibility of three ovarian tumor cell lines (TOV-21G, SKOV-3, and OV-90) to cisplatin and paclitaxel, and to investigate the influence of these treatments on the mRNA expression of TANK, RIPK1, NFKB1, TNFRSF10D, and TRAF2. Among the cell lines, SKOV-3 ovarian adenocarcinoma cells demonstrated the highest resistance to cisplatin treatment (0.125mg/mL), followed by TOV-21G (0.076mg/mL) and OV-90 cells (0.028mg/mL). Regarding paclitaxel treatment, the SKOV-3 cell line exhibited the highest resistance (1.4µg/mL), followed by OV-90 (1.3µg/mL) and TOV-21G cells (0.9µg/mL). Gene expression analysis after paclitaxel treatment remained unchanged; however, after cisplatin treatment, TNFRSF10D was observed to be upregulated nearly 100-fold in SKOV-3 compared to all other cell lines studied. SKOV-3 is described as cisplatin and tumor necrosis factor-resistant. Despite the defective signaling of the TNFRSF10D receptor for apoptosis, it can activate the NFKB transcription factor through non-canonical TRAIL signaling, contributing to a pro-inflammatory immune response. In light of this, damage associated with cisplatin increases TNFRSF10D expression and may promote cell survival through non-canonical NFKB pathway activation. This suggests that resistance to TRAIL-induced apoptosis in these cells could serve as a promising chemoresistance biomarker in OC.

How Progesterone Receptor Expression Impacts Platinum Sensitivity in Ovarian Clear Cell Carcinoma: Insights from Clinical and Experimental Perspectives.

Ovarian clear cell carcinoma (OCCC) is often considered a relatively platinum-resistant malignancy. The aim of this study was to explore the influence of progesterone receptor (PR) expression levels on platinum sensitivity and survival outcomes in people with OCCC. A retrospective analysis was conducted with 80 people with OCCC who underwent surgery followed by adjuvant chemotherapy. PR expression was assessed via immunohistochemical (IHC) staining and quantified using the H score. The platinum sensitivity and survival outcomes of patients with weak and strong PR expression were compared. Additionally, cisplatin viability and migration experiments were conducted with OCCC cell lines (ES-2 and TOV-21G) with varying PR isoform expressions. Among the 80 patients, 62 were classified as having platinum-sensitive disease, while 18 had platinum-resistant disease. The mean total PR H- score of platinum-sensitive tumors was significantly higher than that of platinum-resistant tumors (p = 0.002). Although no significant differences in progression-free and overall survival were observed between patients with high and low PR expression, those with high PR expression tended to have longer survival. While PR protein was only weakly detectable in ES-2 and TOV-21G cells, a transfection of the PR-A or PR-B gene resulted in a strong expression of PR-A or PR-B, which led to significantly reduced proliferation and migration in ES-2 and TOV-21G cells. Furthermore, overexpression of PR-A or PR-B enhanced cisplatin cytotoxicity in these cell lines. In conclusion, strong PR expression was associated with improved platinum sensitivity and survival outcomes, consistent with our experimental findings. The potential of PR as a tumor sensitizer to cisplatin in OCCC warrants further investigation.

Abstract 3240: Synthetic lethal strategy of an EZH1/2 dual inhibitor, HM97662, for the treatment of ARID1A-mutated solid cancers

Abstract Enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of polycomb repressive complex 2 (PRC2), is known to catalyze tri-methylation of histone H3 at lysine 27 (H3K27me3), leading to repression of the transcription of its target genes involved in cell cycle regulation, cell proliferation, cell differentiation, and tumor suppression. It has been proposed that epigenetic regulators could serve as novel drug targets, and EZH2 is one of the targets with considerable therapeutic potential. Meanwhile, synthetic lethality is a phenomenon whereby individual genes in a pair can be knocked-out without affecting cell viability, while disruptions of both genes concurrently cause cell death. It has been shown that loss-of-function (LOF) mutations of component proteins in SWI-SNF chromatin remodeling complex, including PBRM1, ARID1A, SMARCA4, and so on, are synthetic lethal with inhibition of the histone methyltransferase EZH2. Especially, ARID1A-mutated solid tumors are vulnerable to EZH2 inhibition. Given that ARID1A is mutated in approximately 10% of all tumor types, inhibiting EZH2 activity could be a promising therapeutic strategy for cancer patients with this mutation. Although the methyltransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also plays a compensatory role in maintaining tri-methylation of H3K27 and directly binds to chromatin, modulating its condensation. These emphasize that blocking both EZH1 and EZH2 might have greater anti-tumor effect than EZH2 alone. Herein, we introduce a novel EZH1/2 dual inhibitor, HM97662, which concurrently suppressed the methyltransferase activity not only wild-type EZH1, but also wild-type and gain-of-function (GOF) mutant EZH2 at nanomolar concentrations. HM97662 potently repressed tri-methylation of H3K27me3 in TOV-21G (ovarian cancer) and HT-1376 (bladder cancer) cell lines with ARID1A LOF mutation. HM97662 also showed a wide and strong growth inhibitory effect in various solid cancer cell lines harboring ARID1A mutation. HM97662 exhibited potent antitumor activities in xenograft mouse models with various ARID1A-mutated cancer cells including ovarian, bladder, small cell lung cancer, prostate, and gastric cancers (e.g. TOV-21G, HT-1197, NCI-H128, LNCap, and MKN-45, respectively). Once-daily oral dosing of HM97662 greatly inhibited tumor growth in a dose-dependent manner without significant clinical signs compared to known EZH2 selective or EZH1/2 dual inhibitors. HM97662 also exhibited a synergistic effect in combination with second-line standard of care in small cell lung cancer. In conclusion, these preclinical studies demonstrated that HM97662 as an EZH1/2 dual inhibitor, holds promising therapeutic potential for ARID1A-mutated solid cancers. It is crucial to evaluate the effectiveness of HM97662 in subsequent clinical trials. Citation Format: Yu-Yon Kim, Seung Hyun Jung, Seon Yeong Han, Jooyun Byun, Gunwoo Lee, Miyoung Lee, Junghwa Park, Taeyeon Kong, Hyesun Han, Sang Hyun Lee, Young Gil Ahn. Synthetic lethal strategy of an EZH1/2 dual inhibitor, HM97662, for the treatment of ARID1A-mutated solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3240.

Abstract 6441: Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer

Abstract Background. HSF1 helps cancer cells cope with multiple stresses caused by oncogene activation. Methods. See details below. Results. We discovered the bisamide NXP800 as an inhibitor of HSF1-mediated transcription through phenotypic screening and med chem optimization. Gene expression microarray analysis of human cancer cell lines and tumor xenografts treated with bisamide inhibitors indicated activation of the ATF4 axis of the ISR as a key mechanism of action. In SK-OV-3 ovarian cancer xenografts, NXP800 showed clear PK/PD relationships with increased expression of ATF4 transcriptional target genes alongside decreased expression of HSF1 transcriptional targets. NXP800 also caused tumor regressions in this model. Expanding in additional human ovarian cancer xenograft models, we observed efficacy in five and a complete lack of response in three others. The sensitive models all had homozygous deleterious mutations in the ARID1A gene, whereas the non-responding xenografts were all wild type (WT). ARID1A is a component of the SWI/SNF chromatin remodelling complex involved in repression and activation of target genes. Subsequent screening of the large Sanger human cancer cell line panel confirmed ARID1A as the most significant common disease-related alteration predicting sensitivity to NXP800 in ovarian cancer cell lines. This predictive relationship was confirmed in an ARID1A isogenic HCT-116 cell line pair; sensitivity was greater in the homozygous ARID1A mutant cells compared to WT, resulting in PARP cleavage in the mutant cells only. In vivo there was no effect in WT HCT-116 xenografts whereas growth inhibition was observed in the homozygous mutant cells, resulting in significantly smaller tumors. In addition, the induction of ATF4 target genes was stronger and more prolonged in the mutant cells. It is known that whereas short term ATF4 activation is adaptive, persistent activation can promote the induction of apoptosis. CHIP-seq analysis confirmed clear relationships between ARID1A status, ATF4 and HSF1 promoter occupancy, and the distribution of BRG1 and RNA pol II at target sites, although these were often gene-specific and complex. A relatively simple example is the regulation of the INHBE gene (expression of which is a PD biomarker). In untreated samples there is no binding of ATF4, BRG1 and RNA pol II at the INHBE promoter. NXP800 treatment results in the recruitment of BRG1, ATF4 and RNA pol II in ARID1A mutant TOV-21G cells but not in the RMGI WT cells, leading to increased expression in the mutant cells only. Conclusions. We propose that ARID1A loss alters the binding and recruitment of ATF4 and HSF1 leading to the altered and prolonged expression of ATF4 target genes and increased sensitivity to NXP800. NXP800 is currently in phase Ib for the treatment of ARID1A mutant platinum resistant ovarian cancer (NCT05226507). Citation Format: Robert H. te Poele, Marissa Powers, Swee Sharp, Emmanuel de Billy, Maria Taskinen, Loredana Pellegrino, Sharon Gowan, Asadh Miah, Angela Hayes, Matthew Cheeseman, Keith Jones, Suzanne Eccles, Florence Raynaud, Paul Clarke, Paul Workman. Discovery of ARID1A loss as a patient biomarker for NXP800 - A developmental activator of the integrated stress response (ISR) and inhibitor of the HSF1 pathway in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6441.

Transcriptomic analyses of ovarian clear-cell carcinoma with concurrent endometriosis.

Endometriosis, a benign inflammatory disease whereby endometrial-like tissue grows outside the uterus, is a risk factor for endometriosis-associated ovarian cancers. In particular, ovarian endometriomas, cystic lesions of deeply invasive endometriosis, are considered the precursor lesion for ovarian clear-cell carcinoma (OCCC). To explore this transcriptomic landscape, OCCC from women with pathology-proven concurrent endometriosis (n = 4) were compared to benign endometriomas (n = 4) by bulk RNA and small-RNA sequencing. Analysis of protein-coding genes identified 2449 upregulated and 3131 downregulated protein-coding genes (DESeq2, P< 0.05, log2 fold-change > |1|) in OCCC with concurrent endometriosis compared to endometriomas. Gene set enrichment analysis showed upregulation of pathways involved in cell cycle regulation and DNA replication and downregulation of pathways involved in cytokine receptor signaling and matrisome. Comparison of pathway activation scores between the clinical samples and publicly-available datasets for OCCC cell lines revealed significant molecular similarities between OCCC with concurrent endometriosis and OVTOKO, OVISE, RMG1, OVMANA, TOV21G, IGROV1, and JHOC5 cell lines. Analysis of miRNAs revealed 64 upregulated and 61 downregulated mature miRNA molecules (DESeq2, P< 0.05, log2 fold-change > |1|). MiR-10a-5p represented over 21% of the miRNA molecules in OCCC with endometriosis and was significantly upregulated (NGS: log2fold change = 4.37, P = 2.43e-18; QPCR: 8.1-fold change, P< 0.05). Correlation between miR-10a expression level in OCCC cell lines and IC50 (50% inhibitory concentration) of carboplatin in vitro revealed a positive correlation (R2=0.93). MiR-10a overexpression in vitro resulted in a significant decrease in proliferation (n = 6; P< 0.05) compared to transfection with a non-targeting control miRNA. Similarly, the cell-cycle analysis revealed a significant shift in cells from S and G2 to G1 (n = 6; P< 0.0001). Bioinformatic analysis predicted that miR-10a-5p target genes that were downregulated in OCCC with endometriosis were involved in receptor signaling pathways, proliferation, and cell cycle progression. MiR-10a overexpression in vitro was correlated with decreased expression of predicted miR-10a target genes critical for proliferation, cell-cycle regulation, and cell survival including [SERPINE1 (3-fold downregulated; P< 0.05), CDK6 (2.4-fold downregulated; P< 0.05), and RAP2A (2-3-fold downregulated; P< 0.05)]. These studies in OCCC suggest that miR-10a-5p is an impactful, potentially oncogenic molecule, which warrants further studies.

Ribosomal S6 kinase 4 (RSK4) tumor suppressor gene promoter methylation status in ovarian cancer.

Previously, we reported lower RSK4 mRNA and protein levels in malignant ovarian tumors compared to normal and benign ovarian tissues. Also, we observed a significant inverse correlation between the advanced ovarian cancer stages and RSK4 mRNA levels. We did not investigate the mechanisms involved in RSK4-reduced expression in ovarian cancer. Thus, this study investigates whether RSK4 promoter methylation in ovarian cancer tissues is responsible for its low expression. Additionally, the reactivation of RSK4 expression and its effect was studied in ovarian cancer cell lines. RSK4 promoter methylation percentage in malignant and benign ovarian tumors and normal ovary tissues was determined by combined bisulfite restriction analysis. The reactivation of RSK4 expression by decitabine treatment was studied in OVCAR3, SKOV3, TOV-112D, and TOV-21G cells by Western blotting. Cell proliferation was determined by XTT. A significantly high methylation percentage of the RSK4 promoter was observed among malignant and benign ovarian tumors but not in normal ovarian tissue. RSK4 promoter methylation was not associated with age, histological subtype, or stages of ovarian cancer. RSK4 promoter methylation correlates weakly but not significantly with RSK4 protein expression. No correlation was shown between RSK4 methylation and RSK4 mRNA expression. Decitabine induces RSK4 reactivation in all cell lines. However, cell proliferation was reduced only in TOV-112D cells. These data indicate that although RSK4 promoter methylation is increased in malignant ovarian tumors, this mechanism is unlikely to regulate its expression in ovarian cancer. RSK4 reactivation reduced cell proliferation only in the endometroid histological subtype.

Domatinostat Targets the FOXM1-Survivin Axis to Reduce the Viability of Ovarian Cancer Cells Alone and in Combination with Chemotherapeutic Agents.

The deregulation of the FOXM1 transcription factor is a key molecular alteration in ovarian cancer, contributing to the development and progression of ovarian cancer via activation of the target genes. As such, FOXM1 is a highly attractive therapeutic target in the treatment of ovarian cancer, but there has been no clinically tested FOXM1 inhibitor to date. We investigated in this study the effects of domatinostat, a class I-selective HDAC inhibitor currently in the clinical stage of development as a cancer therapeutic, on the expression of FOXM1 and viability of ovarian cancer cells. Cell viability, as well as protein and mRNA expression of FOXM1 and its transcriptional target survivin, was examined after domatinostat treatment of TOV21G and SKOV3 ovarian cancer cell lines in the absence or presence of cisplatin and paclitaxel. The effect of FOXM1 knockdown on survivin expression and those of genetic and pharmacological inhibition of survivin alone or in combination with the chemotherapeutic agents on cell viability were also examined. Domatinostat reduced the protein and mRNA expression of FOXM1 and survivin and also the viability of ovarian cancer cells alone and in combination with cisplatin or paclitaxel at clinically relevant concentrations. Knockdown experiments showed survivin expression was dependent on FOXM1 in ovarian cancer cells. Survivin inhibition was sufficient to reduce the viability of ovarian cancer cells alone and in combination with the chemotherapeutic agents. Our findings suggest that domatinostat, which effectively targets the FOXM1-survivin axis required for the viability of ovarian cancer cells, is a promising option for the treatment of ovarian cancer.

Abstract 5824: Human ovarian cancer cell seeding density as a mechanism of cisplatin resistance associated with expression of anti-apoptotic and autophagy proteins

Abstract Drug effects may vary depending on cancer cell density. We investigated discrepancies in phenotypes for high density and low-density cultured cancer cells and elucidated relevant variables that influence them. We assessed chemosensitivity as a function of tumor cell density by plating TOV-21G and OVMANA ovarian cancer cell lines in 96-well plates at various seeding densities per well and treated them with various concentrations of cisplatin. CellTiterGlo assays were performed at 72 hours of treatment to determine cisplatin dose response curves. We used ethidium homodimer fluorescence staining and imaging to quantify extent of cell death at differing cell densities of TOV-21G and OVMANA cells treated with cisplatin for 48 and 72hours in 48-well plates. We performed western blot analyses of apoptotic and autophagy proteins from TOV-21G and OVMANA cells that were seeded at high and low cell densities in 24-wellplates. TOV-21G and OVMANA cells plated at higher cell seeding densities displayed less cisplatin sensitivity than TOV-21G and OVMANA cells plated at lower seeding densities, as shown by cisplatin dose response curves. Ethidium homodimer staining of TOV-21G and OVMANA cells treated with cisplatin revealed that cells seeded at lower densities experience a greater amount of cell death that cells seeded at higher densities. Western blot analyses showed that high density TOV-21G cells may express greater amounts of BCL2 and c-FLIP and lower amounts of p62 and NOXA compared to low density cells when normalized for protein expression. Similarly, high density OVMANA cells express greater amounts of c-FLIP andBCL2 and lower amounts of NOXA compared to low density cells when normalized for protein expression. Our experiments reveal that human ovarian cancer cells display different phenotypic changes depending on cell density, causing higher density cells to require increased cisplatin to achieve the same level of effect seen in lower density cells. Ovarian cancer cell resistance to cisplatin is associated with alterations in autophagy and anti-apoptotic protein expression at high cell seeding density. These alterations may also affect chemosensitivity in vivo in settings of differential tumor density, such as single circulating tumor cells compared to clumps of tumor cells, and other settings, such as metastasis. Citation Format: Thomas W. Brown, Ujwal Punyamurtula, Wafik S. El-Deiry. Human ovarian cancer cell seeding density as a mechanism of cisplatin resistance associated with expression of anti-apoptotic and autophagy proteins. [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 5824.

Abstract 5512: TIC10/ONC201 in combination with ceralasertib exhibits potent synergy in high grade serous ovarian cancer cells

Abstract High-grade serous ovarian cancer (HGSOC) is the most common and malignant form of ovarian cancer. Although patients with HGSOC are likely to respond to treatment, it is common for the cancer to recur and become resistant to treatment, therefore there is a need to develop new targets and novel combinations to increase patient survival rates. ATRi’s (Ataxia telangiectasia and Rad3 related inhibitors) have shown efficacy in HGSOC. Considering ATRi’s have shown moderate efficacy as a single treatment in HGSOC patients, we hypothesized that combining ATRi’s with ONC201 would enhance efficacy. ONC201 is a pro-apoptotic TRAIL-inducing compound that activates the integrated stress response and inactivates the Akt pathway which is upregulated in over 70% of HGSOC. Inactivation of the Akt pathway may result in a synergistic effect that could be translated for use in patients with HGSOC. Three HGSOC cell lines were treated with the novel drug combination of ceralasertib (ATRi) and ONC201 and CellTiterGLO viability assays were performed after 72 hours to evaluate the synergistic effect of combination treatment. Western blots were performed to investigate the synergistic effect of combination treatment on the Akt pathway as well as expression of the cellular metabolic stress protein ATF4. Cytokine profiling using the Luminex 200 technology was performed to study treatment-induced changes in the tumor microenvironment. Cell viability after ceralasertib and ONC201 combination treatment showed synergy in HGSOC cell lines (OVCAR3, KURAMOCHI, TOV21G). In TOV21G cell line, combination studies of ONC201 and ceralasertib calculated by compusyn software demonstrated a synergistic effect of both drugs, Combination indexes below one were observed at concentration of 0.008-2 μM of ceralasertib with 0.31-5 μM of ONC201 with the best combination index of 0.65. We similarly observed synergy in the two other cell line between ONC201 and ceralasertib. Western blotting showed reduction in total Akt and Erk expression and an increase in ATF4, consistent with the observed synergetic effect of ceralasertib and ONC201 combination. Our ongoing studies are exploring potential mechanisms of synergy between ceralasertib and ONC201 and effects on immune–mediated killing of HGSOC cells. Our results suggest a rational and potentially effective novel therapy combination of ATRi ceralasertib and ONC201 that maybe further developed for HGSOC. Citation Format: Maryam Ghandali, Kadir Mert Selvi, Kelsey E. Huntington, Andrew George, Anna Ochsner, Benedito A. Carneiro, Donn S. Dizon, Wafik S. El-Deiry. TIC10/ONC201 in combination with ceralasertib exhibits potent synergy in high grade serous ovarian cancer cells. [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 5512.

8P NXP800 versus cisplatin in ARID1a-mutated ovarian clear cell carcinoma xenograft models

NXP800 (CCT361814) is a Heat Shock Factor (HSF1) pathway inhibitor currently being studied in a phase 1 clinical trial (NCT05226507). NXP800 demonstrated substantial antitumor activity in cancer cell lines of different histologies, including ovarian clear cell and endometrioid carcinoma (OCCC and EOC), gastric carcinoma, and haematological cancers. Loss of function of ARID1A was identified as a predictive marker for response to NXP800 in OCCC and EOC. Here we describe an in vivo study of NXP800 versus cisplatin as an active control in cisplatin-resistant and cisplatin-sensitive ARID1A-mutated OCCC xenografts, supporting the clinical development of NXP800 in this indication. In vivo studies were performed by generating subcutaneous tumors in Nude (nu/nu) mice using TOV-21G and SKOV3 cell lines. Loss of ARID1A was confirmed in western blot. Treatment with NXP800 resulted in substantial tumor growth inhibition (TGI) in both models. In the TOV-21G model, baseline tumor volumes for the vehicle, cisplatin and NXP800 groups were 201.71, 205.66 and 202.20 mm3, respectively; on Day 28, average tumor volumes were 399.84, 236.68 and 121.47 mm3, respectively, representing increases in tumor volume of 98% for the vehicle control group and 15% for the cisplatin group, and a decrease in tumor volume of 41% for the NXP800 group. In the SKOV3 model, baseline tumor volumes for the vehicle, cisplatin and NXP800 groups were 136.2, 138.5 and 134.7 mm3, respectively; on Day 28, average tumor volumes were 312.94, 253.71 and 53.33 mm3, respectively, representing increases in tumor volume of 130% for the vehicle control group and 80% for the cisplatin group, and a decrease in tumor volume of 60% for the NXP800 group. NXP800 exhibits notable therapeutic activity in xenografts of OCCC – a condition of high unmet medical need with limited treatment options – including sustained tumour growth inhibition and regression both in cisplatin resistant (SKOV3) and sensitive (TOV-21G) OCCC models. Planned phase 1b expansion cohorts will include ovarian clear cell carcinoma and ovarian endometrioid carcinoma patients with ARID1A mutation.

Abstract 1008: Cell density-related variability in chemotherapeutic resistance patterns in human cancer cells

Abstract Assessment of chemosensitivity and resistance varies with cancer cell of origin, drug and culture conditions. However, it is also not uncommon to find multiple different IC50 values for the same chemotherapeutic treatments acting on the same tumor cell lines. The aim of this research was to investigate this discrepancy in phenotypes and elucidate relevant variables that influence them. We assessed chemosensivity as a function of tumor cell density by plating TOV-21G ovarian, HT29 colorectal, and U2OS osteosarcoma cell lines in 96-well plates at various seeding densities per well and treated them with various concentrations of cisplatin, 5-fluorouracil (5-FU), and etoposide, respectively. CellTiter Glo assays were performed at 72 hours of treatment to determine IC50. Cisplatin IC50 for TOV-21G cells plated at 50,000, 20,000, 10,000, 5,000, 2,000, 1,000, and 500 cells/well were 12.00 μM, 5.47 μM, 3.16 μM, 2.45 μM, 2.54 μM, 2.83 μM, and 2.66 μM, respectively. Etoposide IC50 for U2OS cells plated at the same seeding densities were 6.50 μM, 5.19 μM, 3.23 μM, 2.98 μM, 2.49 μM, 2.13 μM, and 1.88 μM, respectively. IC50 values for HT29 cells treated with 5-FU at 2,000, 1,000, and 500 cells/well were 35.89 μM, 19.74 μM, and 13.42 μM, respectively. TOV-21G, U2OS, and HT29 cells all showed differences in IC50 values depending on cell plating density. TOV-21G and U2OS cells both showed a gradual decrease and plateau in IC50 as the cancer cell density decreased from 50,000 cells/well to 500 cells/well. HT29 cells displayed a sharper IC50 decrease as the cell density decreased from 2,000 cells/well to 500 cells/well. Our experiments demonstrate that IC50 values are not static at 72 hours and that tumor cell density is an important variable in influencing observed tumor resistance to traditionally effective drugs. As the density of cancer cells increases, the cells develop more resistance to the associated chemotherapy and require higher quantities of drug to achieve the same level of growth inhibition. We are currently exploring underlying molecular mechanisms that may influence these changes in IC50 including cell survival signaling proteins, cell-cell contact or growth factors that may influence cell density-related chemoresistance. Citation Format: Thomas Brown, Ujwal Punyamurtula, Jill Strandberg, Wafik S. El-Deiry. Cell density-related variability in chemotherapeutic resistance patterns in human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1008.

3P Identification of novel biomarkers of response to ATR inhibitors in ARID1A mutant ovarian clear cell carcinoma

3P Identification of novel biomarkers of response to ATR inhibitors in ARID1A mutant ovarian clear cell carcinoma

The Effect of α-Mangostin and Cisplatin on Ovarian Cancer Cells and the Microenvironment

Ovarian cancer is one of the cancers that, unfortunately, is detected at a late stage of development. The current use of treatment has many side effects. Notably, up to 20% of patients show cisplatin resistance. We assess the effects of cisplatin and/or α-mangostin, a natural plant derivative, on ovarian cancer cells and on the cancer cell microenvironment. The effect of cisplatin and/or α-mangostin on the following cells of ovarian cancer lines: A2780, TOV-21G, and SKOV-3 was verified using the XTT cytotoxicity assay. The separate and combined effects of tested drugs on ovarian cancer cell viability were assessed. We assessed the influence of chemotherapeutic agents on the possibility of modulating the microenvironment. For this purpose, we isolated exosomes from drug-treated and untreated ovarian cancer cells. We estimated the differences in the amounts of exosomes released from cancer cells (NTA technique). We also examined the effects of isolated exosome fractions on normal human cells (NHDF human fibroblast line). In the present study, we demonstrate that treatment of A2780, SKOV-3, and TOV-21G cells with α-mangostin in combination with cisplatin can allow a reduction in cisplatin concentration while maintaining the same cytotoxic effect. Ovarian cancer cells release a variable number of exosomes into the microenvironment when exposed to α-mangostin and/or cisplatin. However, it is important to note that the cargo carried by exosomes released from drug-treated cells may be significantly different.

Cryptocaryone Promotes ROS-Dependent Antiproliferation and Apoptosis in Ovarian Cancer Cells.

Cryptocaryone (CPC) is a bioactive dihydrochalcone derived from Cryptocarya plants, and its antiproliferation was rarely reported, especially for ovarian cancer (OVCA). This study aimed to examine the regulation ability and mechanism of CPC on three histotypes of OVCA cells (SKOV3, TOV-21G, and TOV-112D). In a 24 h MTS assay, CPC showed antiproliferation effects to OVCA cells, i.e., IC50 values 1.5, 3, and 9.5 μM for TOV-21G, SKOV3, and TOV-112D cells. TOV-21G and SKOV3 cells showed hypersensitivity to CPC when applied for exposure time and concentration experiments. For biological processes, CPC stimulated the generation of reactive oxygen species and mitochondrial superoxide and promoted mitochondrial membrane potential dysfunction in TOV-21G and SKOV3 cells. Apoptosis was detected in OVCA cells through subG1 accumulation and annexin V staining. Apoptosis signaling such as caspase 3/7 activities, cleaved poly (ADP-ribose) polymerase, and caspase 3 expressions were upregulated by CPC. Specifically, the intrinsic and extrinsic apoptotic caspase 9 and caspase 8 were overexpressed in OVCA cells following CPC treatment. Moreover, CPC also stimulated DNA damages in terms of γH2AX expression and increased γH2AX foci. CPC also induced 8-hydroxy-2′-deoxyguanosine DNA damages. These CPC-associated principal biological processes were validated to be oxidative stress-dependent by N-acetylcysteine. In conclusion, CPC is a potential anti-OVCA natural product showing oxidative stress-dependent antiproliferation, apoptosis, and DNA damaging functions.

DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the β-Catenin-P-Glycoprotein Signaling Pathway.

Simple SummaryDickkopf-3 (DKK3) is considered a tumor suppressor as it possesses anti-tumoral properties and is frequently downregulated in various cancers. However, the role of DKK3 in ovarian cancer is not known. In this study, we showed that DKK3 loss occurred in 56.1% of patients with ovarian cancer and that it was significantly associated with poor survival and chemoresistance. Secreted DKK3 possessed anti-tumoral properties and enhanced paclitaxel susceptibility by inhibiting the β-catenin-P-glycoprotein signaling pathway in ovarian cancer. This study revealed promising therapeutic effects of secreted DKK3, which targets paclitaxel-resistant ovarian cancer.Dickkopf-3 (DKK3), a tumor suppressor, is frequently downregulated in various cancers. However, the role of DKK3 in ovarian cancer has not been evaluated. This study aimed to assess aberrant DKK3 expression and its role in epithelial ovarian carcinoma. DKK3 expression was assessed using immunohistochemistry with tissue blocks from 82 patients with invasive carcinoma, and 15 normal, 19 benign, and 10 borderline tumors as controls. Survival data were analyzed using Kaplan–Meier and Cox regression analysis. Paclitaxel-resistant cells were established using TOV-21G and OV-90 cell lines. Protein expression was assessed using Western blotting and immunofluorescence analysis. Cell viability was assessed using the MT assay and 3D-spheroid assay. Cell migration was determined using a migration assay. DKK3 was significantly downregulated in invasive carcinoma compared to that in normal, benign, and borderline tumors. DKK3 loss occurred in 56.1% invasive carcinomas and was significantly associated with disease-free survival and chemoresistance in serous adenocarcinoma. DKK3 was lost in paclitaxel-resistant cells, while β-catenin and P-glycoprotein were upregulated. Exogenous secreted DKK3, incorporated by cells, enhanced anti-tumoral effect and paclitaxel susceptibility in paclitaxel-resistant cells, and reduced the levels of active β-catenin and its downstream P-glycoprotein, suggesting that DKK3 can be used as a therapeutic for targeting paclitaxel-resistant cancer.

In vitro assessment for cytotoxicity screening of new antimalarial candidates

In antimalarial research there are no standard procedures to determine the toxicity of a drug candidate. Among the alternatives available, in vitro cytotoxicity assays are the most widely used to predict toxic effects of future therapeutic products. They have the advantage over the in vivo assays, in that they offer the possibility to restrain the number of experimental variables. The objective of the present study was to compare in vitro cytotoxic methods by testing various compounds currently used to treat malaria against different cell lines. Neutral red (NR) uptake and methylthiazoletetrazolium (MTT) colorimetric in vitro assays were used to determine preliminary toxicity of commercially available antimalarial drugs against tumor and non-tumor cells lines. Toxicity through brine shrimp lethality bioassay and hemolytic activity were also evaluated. Significant differences were observed in the tests measured by NR uptake. The tumor cell lines TOV-21G and HepG2 and non-tumor WI-26VA4 cells showed relatively uniform toxicity results, with TOV-21G being the most sensitive cell tested, presenting the lowest concentration to cause death to 50% of viable cells (CC50) values. The results of this study support the use of TOV-21G, HepG2 and WI-26VA4 cells lines as the choice for cytotoxicity tests to evaluate potential bioactive compounds.

Identification of best housekeeping genes for the normalization of RT-qPCR in human cell lines

The identification of the best reference gene is a critical step to evaluate the relative change in mRNA expression of a target gene by RT-qPCR. In this work, we evaluated nineteen genes of different functional classes using Real Time Human Reference Gene Panel (Roche Applied Sciences), to identify the internal housekeeping genes (HKGs) most suitable for gene expression normalization data in human cell lines. Normal cell lines CCD-19LU (lung fibroblast), HEK-293 (epithelial cell of embryonic kidney), WI-26 VA4 (lung fibroblast), and human cancer cells, BT-549 (breast cancer), Hs 578T (breast cancer), MACL-1 (breast cancer), HeLa (cervical carcinoma), U-87 MG (glioblastoma/astrocytoma), RKO-AS45–1 (colorectal carcinoma), and TOV-21G (ovarian adenocarcinoma) were cultivated according to manufacturer’s protocol. Twelve candidate reference genes were commonly expressed in five cell lines (CCD-19Lu, HEK-293, RKO-AS45–1, TOV-21G, and U-87 MG). To verify the expression stability, we used the RefFinder web tool, which integrates data from the computational programs Normfinder, BestKeeper, geNorm, and the comparative Delta-Ct method. The ACTB was the most stable reference gene to the CCD-19Lu and HEK-293 cells. The best combination of HKGs for the RKO-AS45–1 and TOV-21G cell lines were B2M/GAPDH and PBGD/B2M, respectively. For the U-87 MG cells, GAPDH and IPO8 were the most suitable HKGs. Thus, our findings showed that it is crucial to use the right HKGs to precise normalize gene expression levels in cancer studies, once a suitable HKG for one cell type cannot be to the other.

Peritoneal Modulators of Endometriosis-Associated Ovarian Cancer.

Ovarian cancer is the 4th largest cause of cancer death in women. Approximately 10–15% of women of childbearing age suffer from endometriosis. Endometriosis is defined by the growth and presence of endometrial tissue (lesions) outside of the uterus. The women with endometriosis also have an increased presence of peritoneal fluid (PF) that comprises of inflammatory cells, growth factors, cytokines/chemokines, etc. Epidemiological studies have shown that >3% of women with endometriosis develop ovarian cancer (low-grade serous or endometrioid types). Our hypothesis is that the PF from women with endometriosis induces transformative changes in the ovarian cells, leading to ovarian cancer development. PF from women with and without endometriosis was collected after IRB approval and patient consent. IOSE (human normal ovarian epithelial cells) and TOV-21G cells (human ovarian clear cell carcinoma cell line) were treated with various volumes of PF (no endometriosis or endometriosis) for 48 or 96 h and proliferation measured. Expression levels of epigenetic regulators and FoxP3, an inflammatory tumor suppressor, were determined. A Human Cancer Inflammation and Immunity Crosstalk RT2 Profiler PCR array was used to measure changes in cancer related genes in treated cells. Results showed increased growth of TOV-21G cells treated with PF from women with endometriosis versus without endometriosis and compared to IOSE cells. Endo PF treatment induced EZH2, H3K27me3, and FoxP3. The RT2 PCR array of TOV-21G cells treated with endo PF showed upregulation of various inflammatory genes (TLRs, Myd88, etc.). These studies indicate that PF from women with endometriosis can both proliferate and transform ovarian cells and hence this microenvironment plays a major mechanistic role in the progression of endometriosis to ovarian cancer.

Lapatinib and poziotinib overcome ABCB1-mediated paclitaxel resistance in ovarian cancer.

Conventional frontline treatment for ovarian cancer consists of successive chemotherapy cycles of paclitaxel and platinum. Despite the initial favorable responses for most patients, chemotherapy resistance frequently leads to recurrent or refractory disease. New treatment strategies that circumvent or prevent mechanisms of resistance are needed to improve ovarian cancer therapy. We established in vitro paclitaxel-resistant ovarian cancer cell line and organoid models. Gene expression differences in resistant and sensitive lines were analyzed by RNA sequencing. We manipulated candidate genes associated with paclitaxel resistance using siRNA or small molecule inhibitors, and then screened the cells for paclitaxel sensitivity using cell viability assays. We used the Bliss independence model to evaluate the anti-proliferative synergy for drug combinations. ABCB1 expression was upregulated in paclitaxel-resistant TOV-21G (q < 1x10-300), OVCAR3 (q = 7.4x10-156) and novel ovarian tumor organoid (p = 2.4x10-4) models. Previous reports have shown some tyrosine kinase inhibitors can inhibit ABCB1 function. We tested a panel of tyrosine kinase inhibitors for the ability to sensitize resistant ABCB1-overexpressing ovarian cancer cell lines to paclitaxel. We observed synergy when we combined poziotinib or lapatinib with paclitaxel in resistant TOV-21G and OVCAR3 cells. Silencing ABCB1 expression in paclitaxel-resistant TOV-21G and OVCAR3 cells reduced paclitaxel IC50 by 20.7 and 6.2-fold, respectively. Furthermore, we demonstrated direct inhibition of paclitaxel-induced ABCB1 transporter activity by both lapatinib and poziotinib. In conclusion, lapatinib and poziotinib combined with paclitaxel synergizes to inhibit the proliferation of ABCB1-overexpressing ovarian cancer cells in vitro. The addition of FDA-approved lapatinib to second-line paclitaxel therapy is a promising strategy for patients with recurrent ovarian cancer.

Kaempferia parviflora extract inhibits TNF-α-induced release of MCP-1 in ovarian cancer cells through the suppression of NF-κB signaling

Ovarian clear cell carcinoma (OCCC) is an uncommon subtype of epithelial cell ovarian cancers (EOCs) that has poor response to conventional platinum-based therapy. Therefore, finding new potential therapeutic agents is required. Since inflammatory cytokine, tumor necrosis factor alpha (TNF-α), is strongly expressed in EOCs and associated with the level of tumor grade, disruption of this inflammation pathway may provide another potential target for OCCC treatment. We previously reported that Kaempferia parviflora (KP) extract decreased cell proliferation and induced apoptosis. However, the effects of KP on OCCC, especially the aspects related to inflammatory cytokines, have not been elucidated. Our current study demonstrated the effects of KP extract on cytokine production in TNF-α-induced OCCC TOV-21G cell line. This study showed that KP extract inhibited interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production at both transcription and translation levels via the suppression of nuclear factor-kappa B (NF-κB) signal transduction. In contrast, KP extract increased the expression of inhibitor kappa B (IκB) protein which may delay NF-κB translocation into the nucleus upon TNF-α activation. Moreover, the suppression of cytokines released from KP treated-TOV-21G reduced the migration of monocyte cell (THP-1). KP extract also exhibited the inhibition of IL-6 and MCP-1 production from THP-1 activated by lipopolysaccharides (LPS). Cells treated with KP extract exhibited a decrease in extracellular signal-regulated kinases (ERK1/2) and protein kinase B (AKT) phosphorylation and induced myeloid leukemia cell differentiation protein Mcl-1 (MCL-1) expression. Suppression of inflammatory cytokine and chemokine production and inhibition of tumor-associated macrophage (TAM) migration support the possibility of using KP for OCCC treatment.