OVCAR-3 Cell Lines Research Articles

The role of folic acid in inducing of apoptosis by zinc(II) complex in ovary and cervix cancer cells.

Herein, the synthesis, structure, binding affinity, cytotoxicity, and apoptotic properties of the new Zn(II) complex composed of folic acid and bipyridine ligands are reported. Because folic acid has the ability to target cancer cells directly, so it can play a role in targeted drug delivery of the complex and be useful to distinguish normal cells from cancerous. After characterization of Zinc complex utilizing FTIR, EA, and NMR, the results of MTT assay were shown that viability levels of two FR-positive cell lines (HeLa and Ovcar-3) are dependent on time and concentration of [Zn(bpy)FA], whereas, did not show a significant effect on FR-negative cell lines (A549). Also, Real-time PCR revealed that the presence of FA can influence the expression of apoptosis in cervical carcinoma HeLa cells while cisplatin alone doesn't have the ability to trigger apoptosis. Furthermore, the experimental results were evaluated using pharmacophore modeling and molecular docking analysis. Finally, the stability of the Zn(II) complex was surveyed using quantum mechanical studies.

P76RBE silencing inhibits ovarian cancer cell proliferation, migration, and invasion via suppressing the integrin β1/NF-κB pathway

ABSTRACT Rhophilin Rho GTPase binding protein 2 (P76RBE) belongs to rhophilin family of Rho-GTPase-binding proteins and is found to contribute to the development of diverse cancers. Data in Oncomine and Kaplan–Meier Plotter databases showed that P76RBE was upregulated in ovarian cancer tissues compared with normal tissues, and patients with high P76RBE expression had worse overall survival, which indicated P76RBE may be associated with the pathogenesis of ovarian cancer. This study aimed to investigate the role of P76RBE in ovarian cancer and to reveal the possible underlying mechanisms. The results demonstrated that P76RBE was highly expressed in ovarian cancer tissues and ovarian cancer cell lines. Functionally, silencing of P76RBE suppressed the proliferation, induced cell cycle arrest, and inhibited migration and invasion in OVCAR-3 and OV-90 cells, while overexpression of P76RBE showed opposite effects on A2780 cells. Mechanically, P76RBE silencing resulted in downregulation of integrin β1, accompanying the reduced NF-κB p65 phosphorylation and nuclear translocation. Importantly, integrin β1 knockdown effectively rescued the effects of P76RBE overexpression on ovarian cancer cells with suppressed proliferation, migration, and invasion. Additionally, in the xenograft tumors derived from OVCAR-3 and OV-90 cell lines, P76RBE knockdown inhibited tumor growth. Meanwhile, the expression of integrin β1 and NF-κB p65 phosphorylation was decreased. In summary, our findings indicate that P76RBE contributes to the progression of ovarian cancer through regulating the integrin β1/NF-κB signaling, and it may be a promising target for ovarian cancer therapy.

Evaluation of immune responses in a novel ovarian cancer immune-oncology three-dimensional cell culture model

<h3>Objectives:</h3> We aimed to develop and validate a novel ovarian cancer 3D immune oncology co-culture model consisting of high grade ovarian cancer tumor spheroids cultured with immune cells. We evaluated outcomes including tumor spheroid viability, ability to visualize immune cell migration and invasion, cellular expression of immune factors, and cell death resulting from immune co-culture. <h3>Methods:</h3> Spheroids were grown in 96-well U-bottom microplates using SKOV3 and OVCAR8 cell lines. Spheroids were then co-cultured with human peripheral blood mononuclear cells (PBMCs) from gynecologic cancer patients in 96-well 5 µm Transwell plates to allow for immune cell migration and invasion into spheroids. Confocal microscopy of live, labeled cells was performed to visualize migration and spheroid invasion. Flow cytometry was performed to determine spheroid viability over time and expression of immune factors MHC1 and PD-L1 on spheroid cells. Cancer cell death resulting from immune cell co-culture was quantified by propidium iodide immunofluorescence and compared to cell death in spheroids in the absence of PBMC or in spheroids treated with carboplatin. <h3>Results:</h3> SKOV3 and OVCAR8 spheroids became highly compacted after 48-72 hours in culture and retained excellent viability over 1 week. Strong e-cadherin expression was visible in both cell types at 48 hours. Live imaging of spheroids co-cultured with PBMC revealed robust invasion of the spheroids, which increased with increasing PBMC numbers and with time. MHC1 and PD-L1 levels were measured on spheroids alone cultured over 72 hours. Robust expression of MHC1 was observed on SKOV3 cells but was almost absent on OVCAR8 cells. Conversely, prominent expression of PD-L1 was noted on OVCAR8 cells. Immune cell killing of cancer cells was increased compared to controls for both SKOV3 and OVCAR8. OVCAR8 cells demonstrated different dynamics of cell death than SKOV3, with cell death in OVCAR8 peaking at 72 hours and cell death in SKOV3 peaking at 48 hours. <h3>Conclusions:</h3> Our findings demonstrate feasibility and reproducibility of an ovarian cancer 3D immune oncology co-culture model. As compared to traditional 2D tissue culture, this new technique may offer a more physiologic representation of cancer, its microenvironment, and its interaction with the immune system, which may improve preclinical study of cancer and antineoplastic agents.

Effect of Net Charge on DNA-Binding, Protein-Binding and Anticancer Properties of Copper(I) Phosphine-Diimine Complexes

The syntheses of [Cu(PPh3)2(L)]NO3 and [Cu(PPh3)2(L-SO3Na)]NO3 were achieved through the reaction of Cu(PPh3)2NO3 and equimolar amount of the ligands (L = 5,6-diphenyl-3-[2-pyridyl]-1,2,4-triazine; LSO3Na = 5,6-diphenyl-3-[2-pyridyl]-1,2,4-triazine-4,4′-disulfonic acid disodium salt). The complexes were characterized by NMR and IR spectroscopy and mass spectrometry. The compounds exhibit similar absorption and emission spectra, suggesting a similar electronic structure. Ct-DNA binding studies show the strong influence of the net charge as Cu-L (positively charged) is able to bind to ct-DNA while Cu-LSO3Na (negatively charged) is not. The net charge of the complexes affects the thermodynamic and kinetic binding parameters toward human serum albumin. HSA-binding of the complexes was further investigated by molecular docking, revealing different binding sites on the HSA protein as a function of the net charge. The different anticancer activities of the complexes towards ovcar-3 and hope-62 cancer cell lines are suggestive of a role for the overall charge of the complexes. Interaction with the DNA is not the major mechanism for this class of complexes. The overall net charge of the pharmacophore (anticancer agent) should be a key consideration in the design of anticancer metal complexes.

Abstract 2297: Regulatory role of MRCK gamma in ovarian cancer metastasis

Abstract In ovarian cancer, compacted cell aggregates known as spheroids detach from the primary tumor and enable metastasis within the peritoneum. Targeting this ovarian metastatic cascade motivates a delineation of the morphogenetic pathways governing spheroid shedding and invasion. Here we analyze the role of one of three related Myotonic Dystrophy Kinase-Related CDC42-Binding Kinases (MRCKγ) in ovarian cancer metastasis, and test its potential as a therapeutic target. MRCKs are downstream effectors of Cdc42 and Rac1 GTPases and through activation of myosin-II could produce the mechanical force required during cell invasion. MRCKγ is amplified or mutant in 12.5% of 1680 serous ovarian cancer patient samples in TCGA. Analyses of ovarian cancer SKOV3ip cells with 4-fold overexpression of GFP-Rac1 and increased GTPase activation showed a 1.5-fold increase in MRCKγ protein and a concomitant increase in the phosphorylation of myosin required for contractile force generation. A similar increase in myosin phosphorylation was observed in two OVCAR8 cell lines with overexpression of GFP-Rac1 and GFP-Cdc42, respectively. Analyses of the effects MRCKγ knockdown on ovarian spheroid formation and in mesothelial clearance are in progress. To evaluate the therapeutic potential of MRCK/Cdc42BPs we are also testing their pharmacological inhibition with BDP9066 and SD208 during spheroid formation and mesothelial clearance. Our findings are supported by a recent report showing that inhibition of the related MRCKα kinase blocks ovarian cancer cell spheroid formation in vitro. By the end of these studies we will have a better understanding of the role of MRCK/Cdc42BPs in ovarian cancer metastatic pathway, and a potential therapeutic target for ovarian cancer treatment. Citation Format: Leslie Toledo-Jacobo. Regulatory role of MRCK gamma in ovarian cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2297.

Combination treatment with cisplatin, paclitaxel and olaparib has synergistic and dose reduction potential in ovarian cancer cells.

Ovarian cancer is the most lethal type of gynecological cancer. Due to its high heterogeneity and complicated pathological mechanisms, the 5-year survival rate of patients with ovarian cancer is <40%. Tumor cytoreductive surgery and systemic chemotherapy of platinum combined with paclitaxel are currently considered the gold standard for the treatment of ovarian cancer, and chemotherapy resistance has become a key constraint in improving the cure rate of ovarian cancer. Therefore, it is important to identify novel treatment methods and strategies for ovarian cancer. Targeted drugs can not only be used in combination with chemotherapy, but also act as maintenance therapy to promote patient survival time. PARP inhibitor is a novel type of ovarian cancer treatment targeted drug, which can induce an anticancer effect by inhibiting DNA damage and repair of ovarian cancer cells. The present study investigated the different effects of olaparib, cisplatin and paclitaxel in several dosages by single use and combinations on the proliferation of different human ovarian cancer cell lines, in order to verify the synergistic effects of the combinations of the three anticancer agents in pairs. The proliferation inhibitory rate of the cell lines was determined using a Cell Counting Kit-8 assay, while the combination index (CI) value of the combination of three agents in pairs was analyzed using Compusyn software. The proliferation was observed using a crystal violet assay, and the apoptosis ratio was measured via flow cytometry. The results of the present study revealed that the combination of cisplatin with olaparib group had a higher inhibition effect than each single group and had a higher dose-reduction index of >1 than the other two combinations at all concentrations in A2780 and OVCAR-3 cell lines. The difference in proliferation inhibition and induced apoptosis rate of A2780 cell lines was significant in the combination of cisplatin with olaparib group and the control group (P<0.01) at 0.25x IC50. For the OVCAR-3 cell line, the difference was also significant between two groups (P<0.05). The CI values in the A2780 cell line revealed significant differences between the low-dose group (0.0625x, 0.125x and 0.25x IC50) and the high-dose group (0.5x, 1.0x and 2.0x IC50) for the group that received the combination of cisplatin with olaparib (P<0.05). The present study highlighted that the group receiving a combination of cisplatin with olaparib exhibited the most significant synergistic effects among the three combinations, particularly at low doses.

Abstract 1062: Inhibition of ovarian and endometrial cancer cell proliferation by an anti-ROR1 monoclonal antibody

Abstract The non-canonical Wnt signalling receptor ROR1 has been shown to be aberrantly expressed in numerous cancers, including ovarian and endometrial cancer (EC). Cirmtuzumab is a humanised monoclonal antibody against ROR1 that blocks Wnt5a-induced ROR1 signalling. It has demonstrated safety and efficacy in several Phase I/II clinical trials for chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and Her2-negative breast cancer. The aim of this study was to investigate any anti-proliferative effect of cirmtuzumab in combination with commonly-used gynaecological cancer therapies (cisplatin, paclitaxel and the PARP inhibitor, olaparib) on high grade serous ovarian cancer (HGSOC) and EC cell lines including models of platinum resistance. The ROR1 positive HGSOC cells lines CaOV3, CaOV3CisR PEO1, PEO4 and EC cell lines Ishikawa, KLE were used in this study. First, IC50 for cisplatin, paclitaxel and olaparib in each cell line at 72h was determined using the cell counting kit 8 (CCK-8). Then, cells were seeded in 12-well plates and treated with cirmtuzumab at 25µg/ml or 50µg/ml for 4h prior to the addition of the chemotherapeutic agents at IC70 concentration. The effect of cirmtuzumab +/- agents was quantified using the IncuCyte S3 Live Cell Analysis System. Phase contrast cell images were obtained using a 10x objective lens within the instrument every 3h for 72h in total. The average confluence of each well was calculated and normalised against the baseline (time 0). Two-way ANOVA followed by Bonferroni post-test was performed to evaluate the effect of treatments. RNA and protein were extracted at the end of the incubation for qRTPCR and Western blot analysis. Single dose cirmtuzumab at both 25µg/ml and 50µg/ml significantly inhibited proliferation of CaOV3, CaOV3cisR, PEO1 and Ishikawa cells. 50µg/ml of cirmtuzumab decreased proliferation of KLE. Compared to paclitaxel alone, addition of 50µg/ml of cirmtuzumab significantly inhibited proliferation of CaOV3, CaOV3CisR, PEO1 and PEO4. Compared to cisplatin alone, addition of 25µg/ml of cirmtuzumab significantly inhibited proliferation of CaOV3CisR, PEO4; addition of 50µg/ml of cirmtuzumab significantly inhibited proliferation of CaOV3, CaOV3CisR, PEO4. Compared to olaparib alone, addition of 25µg/ml of cirmtuzumab significantly inhibited proliferation of CaOV3CisR; addition of 50µg/ml of cirmtuzumab significantly inhibited proliferation of CaOV3 and CaOV3CisR. No significant change in ROR1 or ROR2 expression levels was observed following cirmtuzumab treatment, however treatment did result in alterations to markers of epithelial-mesenchymal transition (EMT). Cirmtuzumab alone inhibited proliferation of ovarian cancer and EC cells in vitro, and could enhance the activity of commonly-used chemotherapeutic agents. This study supports the potential of cirmtuzumab or other ROR1 targeting therapies for treating women with HGSOC and EC. Citation Format: Dongli Liu, Gunnar F. Kaufmann, James B. Breitmeyer, Kristie-Ann Dickson, Deborah J. Marsh, Caroline E. Ford. Inhibition of ovarian and endometrial cancer cell proliferation by an anti-ROR1 monoclonal antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1062.

Abstract 87: Asparagine synthetase (ASNS) expression predicts response to the GLS1 inhibitor IPN60090 in ovarian cancer through selective modulation of redox homeostasis

Abstract Inhibitors of tumor metabolism have shown promise in the pre-clinical and clinical settings, however success is likely dependent upon identification of responsive patient populations to drive maximum benefit. We have previously disclosed the development of the GLS1 inhibitor, IPN60090, which is currently progressing through Phase 1 studies (NCT03894540). Current efforts are focused on developing additional patient stratification biomarkers to define those patients who will most benefit from IPN60090 single-agent treatment or combination strategies. Here we demonstrate that IPN60090 elicited a specific set of metabolic alterations and selectively inhibited the growth of high grade serous ovarian cancer (HGSOC) models in vitro and in vivo. In IPN60090-sensitive OvCa cell lines, GLS1 inhibition induced glutathione (GSH) depletion, inhibited glutamine anapleurosis (GLN), and altered cell cycle kinetics resulting from depletion of intracellular nucleotide pools and accumulation of DNA damage. Untargeted metabolic profiling of IPN60090-sensitive and -insensitive cell lines revealed that the differential response was driven by the ability of insensitive cell lines to maintain intracellular pools of glutamate (GLU), and consequently GSH, through consumption of aspartate and alanine. We examined two transaminases whose activity may result in aspartate or alanine depletion in cells, asparagine synthetase (ASNS) and glutamate pyruvate transaminase 2 (GPT2), and found that ASNS expression predicted response to IPN60090. In vivo, growth of both subcutaneous and orthotopic ASNSlow OvCa tumors was inhibited by IPN60090, while ASNShigh tumors were resistant to IPN60090. Leveraging tissue microarrays from tumor biopsies collected at MD Anderson Cancer Center, we developed an IHC assay for ASNS to determine the percentage of ASNS null or low patients that would benefit from IPN60090 treatment. Upon validation and CLIA certification, this assay was deployed across archival patient biopsies collected in the Department of Investigational Cancer Therapeutics at MD Anderson Cancer Center, and identified patients who showed no ASNS staining (ASNSnull) in their tumors, suggesting that they may benefit from treatment with IPN60090. Taken together, through a comprehensive translational effort we have identified ASNS as a predictive biomarker of response to GLS1 inhibitor-based therapeutic regimens. Citation Format: Nakia D. Spencer, Christopher A. Bristow, Virginia Giulani, Meredith A. Miller, Alessandro Carugo, Angela L. Harris, Rosalba Minelli, Ningpeng Feng, Qing Chang, Michael J. Soth, Kang Le, John N. Weinstein, Philip L. Lorenzi, Jinsong Liu, Wei-Lien Wang, Timothy A. Yap, Giulio Draetta, Philip Jones, Timothy P. Heffernan, Jeffrey J. Kovacs. Asparagine synthetase (ASNS) expression predicts response to the GLS1 inhibitor IPN60090 in ovarian cancer through selective modulation of redox homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 87.

Abstract 1010: A study of combinatorial growth inhibition, cell death and DNA damage repair caused by CHK1 inhibitor SRA737 and WEE1 inhibitor adavosertib in TP53 mutated cell lines

Abstract Purpose of study: CHK1 and WEE1 are critical determinants of the G2/M cell cycle checkpoint. The G2/M checkpoint is crucial to enable DNA repair caused by endogenous and exogenous stress. We hypothesised that simultaneous inhibition of CHK1 and WEE1 would cause cell death in TP53 mutated cell lines because of the associated replication stress due to an abrogated G1/S checkpoint. Experimental procedures: The potentiation index of the combination of SRA737 and adavosertib was calculated in six cancer cell lines harbouring TP53 mutations (OVCAR3, COV505, BT20, MDA-MB-436 and CAPAN1, MIAPaCa2) using Cell Titre Blue assays. Cell death and DNA damage was quantified using c-PARP, p-CHK1 Ser345 and gamma H2AX using western blot analysis. In-vivo studies evaluated the efficacy of vehicle control, SRA737, adavosertib and the combination in OVCAR3 and MDA-MB-436 xenograft models. We further explored the mechanisms of DNA damage repair caused by the single agents and the combination using two functional fluorescent multiplex DNA repair assay platforms in OVCAR3 and MDA-MB-436 cells; Exy-SPOT (base excision repair/nucleotide excision repair) and Next-SPOT (double strand break repair). Results: The potentiation index of the addition of SRA737 to adavosertib were 19, 8.6, 8.8, 9.7, 27.2 and 5.7 for COV504, OVCAR3, BT20, MDA-MB-436, CAPAN1 and MIAPaCa2 respectively. The combination of both drugs at single agent GI50 concentrations caused increase in c-PARP, p-CHK1 Ser345 and gamma H2AX in all cell lines studied. We chose to explore the combination in the TP53 mutated OVCAR3 and the TP53 and BRCA1 mutated MDA-MB-436 cell lines. In the OVCAR3 xenograft model, the combination of SRA737 and adavosertib caused tumour regressions and tumour volumes were significantly smaller than controls, p&amp;lt;0001. The combination caused a lesser but significant degree of reduction in the tumour volume in the MDA-MB-436 xenograft model compared to control p=0.003. There was less than a 20% loss of mouse body weight in treatment arms of both xenograft models. Functional DNA damage repair analysis showed the combination of SRA737 and adavosertib caused a significantly greater reduction in oxidative stress related base excision repair (Etheno and 8oxoG lesions) as well as double stranded break repair via alternative end joining in the OVCAR3 cell lines compared to MDA-MB-436 cells, p=0.009, p=0.014 and p=0.005 respectively. Conclusions: The combination of SRA737 and adavosertib caused cell death in TP53 mutated cell lines. There were differences in the DNA damage repair mechanisms in across cell lines and the combination showed significant activity in the xenograft models studied. The combination of SRA737 and adavosertib warrants further evaluation for the treatment of selected TP53 mutated cancers. Citation Format: Adam Stewart, Lisa Pickard, Albert E. Hallsworth, Sylvie Sauvaigo, Giovanna Muggiolu, Florence Raynaud, UDAI BANERJI. A study of combinatorial growth inhibition, cell death and DNA damage repair caused by CHK1 inhibitor SRA737 and WEE1 inhibitor adavosertib in TP53 mutated cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1010.

Credentialing and Pharmacologically Targeting PTP4A3 Phosphatase as a Molecular Target for Ovarian Cancer.

High grade serous ovarian cancer (OvCa) frequently becomes drug resistant and often recurs. Consequently, new drug targets and therapies are needed. Bioinformatics-based studies uncovered a relationship between high Protein Tyrosine Phosphatase of Regenerating Liver-3 (PRL3 also known as PTP4A3) expression and poor patient survival in both early and late stage OvCa. PTP4A3 mRNA levels were 5–20 fold higher in drug resistant or high grade serous OvCa cell lines compared to nonmalignant cells. JMS-053 is a potent allosteric small molecule PTP4A3 inhibitor and to explore further the role of PTP4A3 in OvCa, we synthesized and interrogated a series of JMS-053-based analogs in OvCa cell line-based phenotypic assays. While the JMS-053 analogs inhibit in vitro PTP4A3 enzyme activity, none were superior to JMS-053 in reducing high grade serous OvCa cell survival. Because PTP4A3 controls cell migration, we interrogated the effect of JMS-053 on this cancer-relevant process. Both JMS-053 and CRISPR/Cas9 PTP4A3 depletion blocked cell migration. The inhibition caused by JMS-053 required the presence of PTP4A3. JMS-053 caused additive or synergistic in vitro cytotoxicity when combined with paclitaxel and reduced in vivo OvCa dissemination. These results indicate the importance of PTP4A3 in OvCa and support further investigations of the lead inhibitor, JMS-053.

RETRACTED: Tumor-Derived Extracellular Vesicles Promote Activation of Carcinoma-Associated Fibroblasts and Facilitate Invasion and Metastasis of Ovarian Cancer by Carrying miR-630

Ovarian cancer (OC) is a lethal gynecological malignancy. Extracellular vesicles (EVs) are crucial media in cell-to-cell communication by carrying microRNAs (miRs). The current study aims to investigate the underlying mechanism of miR-630 carried by OC cell-derived EVs in regard to invasion and metastasis of OC cells. miRs related to OC metastasis were searched and screened. The expression patterns of screened miRs in human normal fibroblasts (NFs) and carcinoma-associated fibroblasts (CAFs) were detected using RT-qPCR. miR-630 related to OC metastasis and CAFs activation was analyzed further. The levels of FAP and α-SMA were detected using Western blotting and immunofluorescence. The migration of NFs was measured using Transwell assay. OC cell-derived EVs were isolated and identified. Uptake of EVs by NFs was observed using immunofluorescence staining. The culture supernatant of NFs was collected and used to culture the low metastasis cell line OVCAR8. The migration and invasion of OC cells and epithelial mesenchymal transition (EMT) were measured. Moreover, a xenograft model was established by injecting OVCAR8 cells of different groups into nude mice. Lastly, the effect of EV-pretreated NFs on invasion and metastasis of OC cells was observed in vivo. miR-630 was upregulated in OC cells and CAFs, and further associated with CAF activation and OC metastasis. miR-630 overexpression increased the levels of FAP and α-SMA in NFs, resulting in the transformation of NFs into CAFs. EVs carried miR-630 into NFs and EVs promoted CAF activation. miR-630 targeted KLF6. miR-630 inhibition or KLF6 overexpression attenuated EVs-induced CAF activation. EVs activated the NF-κB pathway via the miR-630/KLF6 axis. The conditioned medium of NFs pretreated with EVs promoted the invasion and metastasis of OVCAR8 cells, while downregulating miR-630 in EVs partially inhibited the promotive effect of NFs. EV-pretreated NFs promoted invasion and metastasis of OC in vivo. In conclusion, EVs carried miR-630 into NFs, thereby facilitating CAF activation and promoting invasion and metastasis of OC by inhibiting KLF6 and activating the NF-κB pathway. Our findings might offer a novel mechanism of invasion and metastasis of OC from the perspective of tumor microenvironment.

MiR-198 inhibits proliferation, invasion and migration of ovarian cancer cells by regulating the PI3K/Akt signaling pathway.

The specific objective of this investigation is to explore the impact of miR-198 on proliferation, migration as well as invasion of ovarian cancer (OC) cells. OC tissue and adjacent normal tissue samples from OC patients were collected, and normal human ovarian epithelial cell IOSE80 and OC cell lines SKOV3, Caov3, A2780 and OVCAR3 were selected in this study for investigation. MiR-198 expression level was assessed using RT-qPCR. MTT, colony formation assay, Transwell and wound healing assay, and flow cytometry were adopted to analyze the role of miR-198 in OVCAR cell proliferation, invasion, migration, as well as apoptosis. Meanwhile, the levels of P13K/Akt signaling pathway-related proteins were determined by western blotting. A significant decrease in miR-198 level was revealed in the OC tissues and cells, contributing to the promotion of OVCAR3 cells in terms of proliferation, migration, invasion, and inhibition of apoptosis. MiR-198 overexpression had an opposite effect on these biological processes of OVCAR3 cells. Further study found that down-regulation of miR-198 caused a significant increase in the activity of PI3K/Akt signaling pathway in the OVCAR3 cells. In contrast, overexpressed miR-198 led to inhibition of this pathway's activity. MiR-198 may possess an ability to inhibit activation of the P13K/Akt pathway, thus suppressing the OC cell proliferation, migration, as well as invasion.

The Enhanced Efficacy of Intracellular Delivery of Doxorubicin/C6-Ceramide Combination Mediated by the F3 Peptide/Nucleolin System Is Supported by the Downregulation of the PI3K/Akt Pathway.

Simple SummaryTargeted nanomedicine-based approaches that aim at the simultaneous delivery of synergistic drug combinations to multiple cellular populations are of high relevance for tackling heterogeneity on solid tumors. Considering that cancer stem cells (CSC) may originate from non-stem cancer cells, single-drug regimens targeting only one of these cell populations could enable tumors to evade treatments. As such, the identification of a common marker, such as nucleolin, might result in a therapeutic advantage. The results herein generated suggested a transversal role of nucleolin in the internalization of F3 peptide-targeted pegylated pH-sensitive liposomes into bulk ovarian cancer cells, including putative CSC-enriched ovarian cells. The intracellular delivery of a drug combination such as the one tested herein was relevant in the context of cell lines with higher intrinsic resistances to doxorubicin. The enhanced efficacy of the F3 peptide-targeted liposomal combination of doxorubicin/C6-ceramide was supported by the downregulation of the Akt pathway, within a specific range of basal level of expression.Targeting multiple cellular populations is of high therapeutic relevance for the tackling of solid tumors heterogeneity. Herein, the ability of pegylated and pH-sensitive liposomes, functionalized with the nucleolin-binding F3 peptide and containing doxorubicin (DXR)/C6-ceramide synergistic combination, to target, in vitro, ovarian cancer, including ovarian cancer stem cells (CSC), was assessed. The underlying molecular mechanism of action of the nucleolin-mediated intracellular delivery of C6-ceramide to cancer cells was also explored. The assessment of overexpression of surface nucleolin expression by flow cytometry was critical to dissipate differences identified by Western blot in membrane/cytoplasm of SKOV-3, OVCAR-3 and TOV-112D ovarian cancer cell lines. The former was in line with the significant extent of uptake into (bulk) ovarian cancer cells, relative to non-targeted and non-specific counterparts. This pattern of uptake was recapitulated with putative CSC-enriched ovarian SKOV-3 and OVCAR-3 sub-population (EpCAMhigh/CD44high). Co-encapsulation of DXR:C6-ceramide into F3 peptide-targeted liposomes improved cytotoxic activity relative to liposomes containing DXR alone, in an extent that depended on the intrinsic resistance to DXR and on the incubation time. The enhanced cytotoxicity of the targeted combination was mechanistically supported by the downregulation of PI3K/Akt pathway by C6-ceramide, only among the nucleolin-overexpressing cancer cells presenting a basal p-Akt/total Akt ratio lower than 1.

Synthesis, Structural Studies, and Anticancer Properties of [CuBr(PPh3)2(4,6-Dimethyl-2-Thiopyrimidine-κS

CuBr(PPh3)2(4,6-dimethylpyrimidine-2-thione) (Cu-L) was synthesized by stirring CuBr(PPh3)3 and 4,6-dimethylpyrimidine-2-thione in dichloromethane. The crystal structure of Cu-L was obtained, and indicated that the complex adopts a distorted tetrahedral structure with several intramolecular hydrogen bonds. Moreover, a centrosymmetric dimer is formed by the intermolecular hydrogen bonding of the bromine acceptor created by symmetry operation 1−x, 1−y, 1−z to the methyl group (D3 = C42) of the pyrimidine–thione ligand. HSA-binding of Cu-L and its ligand were evaluated, revealing that Cu-L binds to HSA differently than its ligand. The HSA-bindings were modeled by molecular docking, which suggested that Cu-L binds to the II A domain while L binds between the I B and II A domains. Anticancer activities toward OVCAR-3 and HeLa cell lines were tested and indicated the significance of the copper center in enhancing the cytotoxic effect; negligible toxicities for L and Cu-L were observed towards a non-cancer cell line. The current study highlights the potential of copper(I)-phosphine complexes containing thione ligands as therapeutic agents.

Evaluation of antioxidant and anticancer effects of Lavandula angustifolia and Melissa officinalis on HeLa, OVCAR-3 and MCF-7 cancer cell lines

Evaluation of antioxidant and anticancer effects of Lavandula angustifolia and Melissa officinalis on HeLa, OVCAR-3 and MCF-7 cancer cell lines

P53 Gain-of-Function Mutants and Steroids in Ovarian Cancer Cell Metastasis

High-grade serous ovarian cancer (HGSOC) is a heterogeneous disease for which there currentlyis no cure. Because p53 is mutated in >90% of all ovarian cancer, we studied specific gain-of-function (GOF) p53 mutants and steroid hormones for tumor morphology and metastasis in vivo. For this, we analyzed ALST (WT p53), SKOV3 (p53 null), TYK-NU (p53-R175H), OVCAR3 (p53-R248Q) and OVCA420 (p53-R273H) cell line xenografts in Foxn1-/- mice. ALST cells failed tometastasize, likely due to the known apoptotic effects of WT p53. SKOV3 and the p53-GOF celllines metastasized to the omentum and exhibited distinct morphologies: SKOV3, epithelial-like;TYK-Nu, vascular-like; OVCAR3, epithelial/mesenchymal; and OVCA420 epithelial exclusive. Despite different morphologies and p53 status, each tumor type contained large, Polyploid GiantCancer Cells (PGCCs) that are stem-like cells undergoing endoreplication. A specificphosphorylated, active form of β−catenin (pCTNNB1-S31/S37/T41; pCTNNB1) co-localizedselectively with GOF p53 and the mitotic stress regulatory kinase pMSK1-T581 in mitotic cells andPGCCs, indicating that in addition to GOF p53 mutants, pCTNNB1 and pMSK1 play a role intumor progression. To determine if ALST cells could be rendered metastatic, the p53 GOFmutants R175H and R273H were stably expressed in these cells. Remarkably, the ALST(WT/R273H) cells, but not the ALST (WT/R175H) cells, formed solid tumors on the ovary, visceralfat and uterus; but not on the omentum where OVCA420 (p53-R273H) cells formed tumors. TheALST (WT/R273H) tumors harbored discreet populations of pCTNNB1+ mitotic cells and PGCCsand exhibited distinct growth-promoting responses to estradiol while showing growth-inhibitoryeffects of DHT and nuclear AR in the tumor-associated stromal cells. Stably expressing p53-R175H or p53-R273H GOF mutants in SKOV3 (p53 null) cells enhanced tumor progression withthe R273H mutant being most aggressive. The parental SKOV3 tumors also contained pCTNNB1that was associated with mitotic cells and PGCCs. In the SKOV3 p53-R175H cells - and moreimpressively in the p53-R273H cells - pCTNNB1 co-localized with p53 in PGCCs. Whereas ESR1staining was diffuse in the ALST-R273H cells, it was nuclear in the SKOV3 cell lines. However,tumor promotion by estrogen and inhibition by DHT were observed in each SKOV3 cell line,suggesting that stromal cells may also contribute to the steroid dependent effects. Collectively, 1)elevated levels of specific phosphorylated forms of CTNNB1 and MSK1 identify mitotic cells andPGCCs within the tumors and new targets for therapeutic approaches; 2) knowing the status ofp53, and the presence and localization of pCTNNB1 and pMSK1 and steroid hormone receptorsin ovarian tumors suggest combinatorial approaches should be considered to combat the mostlethal gynecological cancer. NIH-CA181808; NIH-HD097321 (JSR).

Targeting Interleukin‐1 Receptor‐Associated Kinase 1 (IRAK1) In Ovarian Cancer

Ovarian cancer (OvCa) is the leading cause of gynecologic cancer-related deaths, due largely to late-stage diagnoses and the development of chemoresistant disease. Nearly 70% of affected women develop recurrent disease, characterized by resistance to standard chemotherapy despite initial response. Cancer stem cells (CSCs) are a subset of cells that have an innate ability to self-renew and enter a dormant state and have been further implicated in resistance to chemotherapy due to high expression of the ATP-binding Cassette (ABC) transporters. To understand the pathways that may be implicated in enhancing this stem-like phenotype, RNA sequencing was performed on cisplatin sensitive and isogenic cisplatin-resistant OvCa cell lines, as well as cells grown in 2- dimensional (2-D) or 3-dimensional (3-D) cultures. OvCa cells grown as spheroids, a 3-D growth model, are enriched with CSCs, and therefore represent a surrogate for stemness. Interestingly, the Toll-like (TLR) or interleukin-1 (IL-1) receptor pathways were highly enriched in resistant cancer cells and spheroid cultures. A critical factor in these signaling pathways is the interleukin receptor associated kinase 1 (IRAK1). IRAK1 is also highly expressed in chemoresistant OvCa and is responsible for activating numerous downstream pathways involved in tumor progression, such as NF-kB, P38/MAPK, and C-Jun. High expression of IRAK1 is associated with poor overall survival based on analysis of the Cancer Genome Atlas (TCGA) publicly available datasets. Due to the important role IRAK1 has in OvCa growth signaling and patient outcomes, IRAK1 represents an attractive target for CSC-targeted drug therapy. The present study is aimed at identifying small molecule inhibitors of IRAK1 by using in-silico molecular docking-based screening. FDA-approved compound libraries were screened using Autodock Vina software. Compounds were assessed for binding within the c-terminal kinase domain. The compounds are ranked-based on their binding energies (BE) and hydrogen bonding patterns. Our screen identified multiple compounds, one of which included mebendazole, an anthelminthic with a BE of -10.3 kCal/mol. This suggests potent interaction with the kinase domain of IRAK1. To confirm the binding in vitro, we performed cellular thermal shift stability assays (CETSA). We observed that mebendazole treatment protected IRAK1 protein from thermal denaturation, confirming the in silico docking data. Lastly, we performed hexosaminidase proliferation assays to assess the effectiveness of mebendazole on inhibiting cancer cell growth. Cisplatin sensitive OvCar3, A1847, A2780 OvCa and cisplatin resistant C30 OvCa cell lines were treated with increasing doses of mebendazole. The inhibitory concentration 50 (IC50) was determined to be approximately 300 nM at 72h for all cell lines. This suggests that mebendazole has potent anti-cancer activity that may be, in part, mediated through the inhibition of IRAK1.

Evaluating the Inhibition of the Na + ‐H + exchanger Isoform 1 (NHE1) and Kinases that Activate NHE1 as a Potential Combination Therapies for the Treatment of Ovarian Cancer with Paclitaxel

Every year in the United States there are over 22,000 newly diagnosed cases of ovarian cancer leading to over 14,000 deaths. Ovarian Cancer is the fifth leading cause of cancerous deaths among women. Due to that advanced stage of cancer progression at the time of diagnosis treatment is typically challenging and frequently accompanied by drug resistance and disease recurrence. Increased expression and activation of NHE1 has been shown to regulate cell proliferation in cancer cells from ovarian cancer patients. NHE1 is an 815 amino acid transmembrane protein that regulates intracellular pH. NHE1 hyperactivity is involved in the pH inversion which creates an optimal environment for cancer cell proliferation and invasion. In an effort to combat this disease recurrence and drug resistance, it is hypothesized that combining the chemotherapeutic paclitaxel with inhibitors that alter NHE1 function there will be a synergistic effect that slows ovarian cancer progression. Paclitaxel is a secondary treatment for ovarian cancer. It is a taxane based chemotherapeutic that acts as a microtubule stabilizer that affects the cells in the G2 and M phases of cell growth. In these experiments a direct inhibitor of NHE1, Ethylisopropylamiloride, is used. Additionally, we evaluate BI-D1870, the inhibitor of Ribosomal S6 Kinase (Rsk). Rsk is activated in ovarian cancer and has the ability to stimulate NHE1 activity. The ability to develop a synergistic effect was assessed using a proliferation assay in two ovarian cancer cell lines CAOV-3 and SKOV-3. Proliferation assays were run with paclitaxel alone and then with both paclitaxel and one of the inhibitors. The IC50 values were then determined for paclitaxel alone and in combination with the inhibitor. For example, in SKOV-3 Cells, the IC50 for paclitaxel was 0.086 µM. When combined with BI-D 1870 the IC50 for paclitaxel decreased to 0.12 µM, an 86% reduction. These data indicate that inhibitors of NHE1 or NHE1 activation may be combined with paclitaxel to improve the efficacy of ovarian cancer treatment.

PLODs are overexpressed in ovarian cancer and are associated with gap junctions via connexin 43

Procollagen-lysine, 2-oxoglutarate 5-dioxygenases (PLODs) play important roles in cancer progression, but their role in ovarian cancer remains elusive. In silico analysis of expression of PLODs in ovarian cancer was performed with reproduction of The Cancer Genome Atlas dataset. PLOD-enriched pathways and related gene(s) were validated by immunohistochemistry (IHC) in 80 ovarian cancer tissue blocks and in vivo xenograft murine models. PLODs (PLOD-1, -2, and -3) were overexpressed in ovarian cancer tissue. Overexpression of individual PLODs showed mutual exclusivity. Each of the three PLODs was differentially expressed between normal and cancer tissue of the ovary. PLOD1 was not prognostic, whereas lower PLOD2 and higher PLOD3 expression were associated with worsened prognosis, respectively. Cases with PLOD overexpression showed enrichment in gap junctions. GJA1 (connexin 43) was significantly overexpressed in cases with PLOD overexpression. IHC in tissue showed the strongest positive correlation between PLOD3 and connexin 43 expression, followed by PLOD2. As per Harmonizome, we selected SKOV3 and CAOV3 cell lines based on constitutive high PLOD1 and PLOD2/PLOD3 expression, respectively for in vitro and in vivo modeling. Only knockdown of PLOD3 was significantly associated with decreased GJA1 expression level in both cell lines. IHC in murine xenograft tumors also showed significantly lower connexin 43 in PLOD3-KD SKOV3 tumors. We conclude that PLODs are generally overexpressed in ovarian cancer and each PLOD may be functionally non-redundant. Association between PLOD3 and gap junctions warrants further investigation.

RHPN1‑AS1 promotes ovarian carcinogenesis by sponging miR‑485‑5p and releasing TPX2 mRNA.

Long non-coding RNAs (lncRNAs) play a crucial role in cancer development. However, researchers have yet to identify the underlying association between lncRNAs and ovarian cancer (OC). The aim of the present study was to examine the effect of lncRNA RHPN1-AS1 (RHPN1-AS1) on OC cells and tissues. Reverse transcriptase-quantitative PCR (RT-qPCR) was utilized to quantify RHPN1-AS1, miR-485-5p, and TPX2 mRNA expression in samples with OC. Luciferase-reporter assay, RNA immunoprecipitation (RIP) assay, and RNA pull-down assay were then employed to validate the target relationship among RHPN1-AS1, miR-485-5p and TPX2. Cell Counting Kit-8, BrdU, wound-healing, cell-adhesion, and flow cytometry assays were also employed to assess cell viability, proliferation, migration, adhesion and apoptosis, respectively, in SKOV3 and OVCAR3 cell lines. Findings revealed that RHPN1-AS1 demonstrated a higher expression level in OC cell lines and tissues. In addition, RHPN1-AS1 enhanced the adhesion, proliferation and migration of OC cell lines but decreased apoptosis of OC cells. It was also observed that the relationship between RHPN1-AS1 and miR-485-5p was negative and that RHPN1-AS1 could sponge miR-485-5p to regulate the proliferation, apoptosis, adhesion, and migration abilities of OC cells. Moreover, TPX2 was targeted by miR-485-5p and was significantly overexpressed in OC cell lines and tissues. Experimental investigations also revealed that TPX2 promoted the proliferation, adhesion, and migration of OC cells but suppressed the apoptosis of SKOV3 and OVCAR3 cells. In summary, RHPN1-AS1 played a tumor promotive role by sponging miR-485-5p to increase TPX2 expression in OC tumorigenesis.