SKOV3 Cell Lines Research Articles

MicroRNA-33b replacement effect on growth and migration inhibition in ovarian cancer cells.

Ovarian cancer is a devastating gynecological disease which is considered the major cause of cancer fatality around the world. The down-regulation of microRNA-33b (miR-33b) was reported in some malignancies. Hence, we transfected the miR-33b mimic into SKOV3 cells and evaluated the impacts of this interference on the growth and migration repression of these tumor cells as well as on targeted genes expression. In our study, transfecting the miR-33b mimic and inhibitor, negative control (NC), and NC inhibitor were established using Lipofectamine 2000. The cytotoxic effects of miR-33b were evaluated by MTT. To assess the miR-33b effects on cell migration, a scratching test was applied. The expression levels of miR-33b, ADAMTS, C-Myc, MMP9, K-Ras, and CXCR4 were evaluated using qRT-PCR. These findings indicate that transfection of miR-143mimic had no marked effects on the SKOV3 cell line. As expected, miR-33b relative expression levels were as follows: miR-33b mimic >NC and NC inhibitor >miR-33b inhibitor (p<0.01). Moreover, transfected miR-33b mimic could suppress SKOV3 cells' proliferation, whereas transfected miR-33b inhibitor could promote cell proliferation (p<0.01). MiR-33b overexpression significantly down-regulated the MMP9, CXCR-4, c-Myc, ADAMTS, and K-Ras mRNA levels (p<0.05). As expected, these results confirm the tumor-suppressive effect of miR-33b in the SKOV3 ovarian cancer cell line by reducing cell survival, proliferation, and migration.

LINC00035 Transcriptional Regulation of SLC16A3 via CEBPB Affects Glycolysis and Cell Apoptosis in Ovarian Cancer.

Objective Ovarian cancer (OC) represents the most lethal gynecologic malignancy globally. Over the decades, lncRNAs have been considered as study focuses due to their genome-wide expression through multiple mechanisms in which regulation of target gene transcription through interaction with transcription factors or epigenetic proteins is proven. In the present work, we focus on the functional role of LINC00035 in OC and its regulation mechanism on gene expression. Methods We collected OC tissues and adjacent tumor-free tissues surgically resected from 67 OC patients. Cultured human OC cell lines SKOV3 and A2780 were assayed for their viability, migration, invasion, apoptosis in vitro using CCK-8 assays, transwell assays, and flow cytometric analysis. OC cell tumorigenesis in vivo was evaluated by mouse xenograft experiments. Glycolysis was evaluated by glucose uptake, lactate release, and ATP production assays. Luciferase activity assay, RNA immunoprecipitation (RIP), and RNA pull-down were performed to confirm the interactions among LINC00035, CEBPB, and SLC16A3. Results LINC00035 was upregulated in OC tissues. LINC00035 knockdown was shown to repress SKOV3 and A2780 cell viability, migration, invasion, induce their apoptosis, and reduce glucose uptake, lactate release, and ATP production. LINC00035 could recruit CEBPB into the SLC16A3 promoter region, thus increasing the SLC16A3 transcription. SLC16A3 was upregulated in OC tissues. SLC16A3 knockdown exerted similar effects on SKOV3 and A2780 cells as LINC00035 knockdown. Rescue experiments found SLC16A3 overexpression resisting to LINC00035 knockdown on SKOV3 and A2780 cell viability, migration, invasion, apoptosis, glucose uptake, lactate release, and ATP production. Results also showed LINC00035 knockdown could inhibit OC cell tumorigenesis in vivo. Conclusion The study reveals that LINC00035 promotes OC progression by regulating glycolysis and cell apoptosis through CEBPB-mediated transcriptional promotion of SLC16A3.

Application of Bioinformatics Analysis to Identify Important Pathways and Hub Genes in Ovarian Cancer Affected by WT1.

Wilms tumor gene (WT1) is used as a marker for the diagnosis and prognosis of ovarian cancer. However, the molecular mechanisms involving WT1 in ovarian cancer require further study. Herein, we used bioinformatics and other methods to identify important pathways and hub genes in ovarian cancer affected by WT1. The results showed that WT1 is highly expressed in ovarian cancer and is closely related to the overall survival and progression-free survival (PFS) of ovarian cancer. In ovarian cancer cell line SKOV3, WT1 downregulation increased the mRNA expression of 638 genes and decreased the mRNA expression of 512 genes, which were enriched in the FoxO, AMPK, and the Hippo signaling pathways. The STRING online tool and Cytoscape software were used to construct a Protein-protein interaction (PPI) network and for Module analysis, and 18 differentially expressed genes (DEGs) were selected. Kaplan-Meier plotter analysis revealed that 16 of 18 genes were related to prognosis. Analysis of GEPIA datasets indicated that 7 of 16 genes were differentially expressed in ovarian cancer tissues and in normal tissues. The expression of IGFBP1 and FBN1 genes increased significantly after WT1 interference, while the expression of the SERPINA1 gene decreased significantly. The correlation between WT1 expression and that of these three genes was consistent with that of ovarian cancer tissues and normal tissues. According to the GeneMANIA online website analysis, there were complex interactions between WT1, IGFBP1, FBN1, SERPINA1, and 20 other genes. In conclusion, we have identified important signaling pathways involving WT1 that affect ovarian cancer, and distinguished three differentially expressed genes regulated by WT1 associated with the prognosis of ovarian cancer. Our findings provide evidence outlining mechanisms involving WT1 gene expression in ovarian cancer and provides a rational for novel treatment of ovarian cancer.

Cytotoxic effect of Tilia dasystyla and Polygonatum orientale Desf extracts on AGS and SKOV-3 cancer cell lines

Nowadays medicinal plants have been considered as the complementary medicine in cancer treatment by researchers. Some plants possess chemical compounds which are able to inhibit the growth of cancer cells or even eliminate them through apoptosis or necrosis. In current study anticancer effect of Tilia dasystyla and Polygonatum orientale Desf extracts on AGS and SKOV-3 cancer cell lines were investigated. The cytotoxic effect of Tilia dasystyla and Polygonatum orientale Desf extracts on AGS and SKOV-3 has not been reported so far. Cancer cell lines were treated with different concentrations (100-5000 µg/ml) of T. dasystyla and P. orientale Desf methanol extracts for 24, 48, 72 hours. Cell viability of AGS and SKOV-3 cancer cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method. Results of the study indicate that, extracts of T. dasystyla and P. orientale Desf showed cytotoxic effect on AGS and SKOV-3 cancer cell lines. The lowest IC50 value of AGS and SKOV-3 cell lines was about 1.02 ± 0.01 mg /ml and 1.14 ± 0.17 mg / ml respectively. T. dasystyla and P. orientale Desf extracts showed cytotoxic effect on AGS and SKOV-3 cancer cell lines in time- and dose-dependent manner. Full potential of the extracts, as an option for cancer treatment, is yet to be determined by further studies on animal models and subsequent trials.

Evaluation of Cytotoxicity and Molecular Docking Studies of Phthalimide and Naphthalimide Derivatives as Potential Anticancer Agents

Background and Objectives Cancer is the second leading cause of death in the world after cardiovascular disease, so the aim of the present study was to investigate phthalimide and naphthalimide derivatives in order to develop anticancer compounds. Subjects and Methods In this study, the cytotoxic activity of six phthalimed and naphthalamide derivatives was evaluated using MTT method on three cancerous cell lines, including breast cancer (MCF-7), ovarian cancer (SKOV3) and lung cancer (A549) cell line. Molecular Docking studies were also performed to determine the binding energy and the compounds interaction with DNA as a possible target of these compounds. Results Based on MTT results, compound C1, a naphthalimide derivative, showed the highest cytotoxic activity. IC50 values of this compound against MCF-7, SKOV3 and A549 cancer cell lines were 1.7, 6.2 and 9.5 μM, respectively. Also, comparison of phthalimide and naphthalimide derivatives showed that compounds C1, C3, C5 with carboxyl group had better effects than other compounds, C2, C6, C4, which bearing 5-amidoisophthalic acid moiety. Conclusion In general, naphthalimide derivatives showed better cytotoxicity than phthalimide derivatives. Compound C1 has the highest cytotoxic activity on all three cancer cell lines and can be further studied in the development of new anti-cancer compounds.

P53-Induced Autophagy Regulates Chemotherapy and Radiotherapy Resistance in Multidrug Resistance Cancer Cells.

BackgroundMultidrug resistance (MDR), a major problem in oncology therapy, limits the effectiveness of anticancer drugs. Although p53 functions as a tumor suppressor, the associations between p53 status, autophagy, and MDR are complicated and conditional.Method In this report, p53-null human ovarian cancer cell line SKOV3 and its MDR phenotype SKVCR and human leukemia cell line CEM and its MDR phenotype CEM-VLB) (p53 mutant cell line) were used.Results Compared to parental SKOV3, the mRNA and protein levels of MAPLC3-II and Beclin1 were higher in SKVCR cells. The inhibition of autophagy by 3-MA significantly sensitized SKVCR to VCR. Conversely, in drug-resistant leukemic cells CEM-VLB, the expressions of Beclin1 and MAPLC3-II were lower than CEM. CEM and CEM-VLB cells were treated with VLB .01 or 0.5 μg/mL, respectively, and the expression of p53 and autophagy up-regulated after VLB (.01 μg/mL) treatment in CEM cells. The percentage of S-phase and G2/M phase cells up-regulated significantly by .01 μg/mL VLB in CEM, which may relate to the status of p53 of CEM cells. A combination of radiation with 3-MA significantly increased apoptosis in CEM-VLB cells.Conclusion Our discovery found that p53 is an important regulator controlling the balance between autophagy and MDR, as a potential drug target for ovarian cancer and leukemia.

Synthesis and evaluation of SPION@CMD@Ser-LTVSPWY peptide as a targeted probe for detection of HER2+ cancer cells in MRI

Objective(s): Successful detection of tumors in the early stages can significantly increase a patient’s healing process and recovery speed. Conventional imaging techniques usually depend on the tissues’ anatomical structure. Epidermal growth factor receptor-2 (HER-2) is a transmembrane protein with an extracellular ligand-binding domain. HER2 plays an essential role in cell proliferation, differentiation, and survival, and its overexpression is associated with various cancers, especially breast and ovarian cancers. Access to its extracellular domain makes HER2 an ideal target for drug preparation and molecular imaging probes. In this study, a targeted magnetic nanoprobe for molecular imaging of HER2 positive cancers was synthesized, and also its potential as a T2-weighted targeted contrast agent was assessed.Materials and Methods: Superparamagnetic SPION nanoparticles were synthesized using the co-precipitation method in the presence of CMD and were labeled with SLTVSPWY peptide. The SPION@CMD@SLTVSPWY nanocomplex was characterized by TEM, DLS, XRD, AAS, FTIR, EDX, and VSM. The r1 and r2 relaxivities were then calculated using a 1.5 Tesla clinical magnetic field. The cytotoxicity of the nanocomplex was evaluated by MTT assay. Finally, the difference between uptake of targeted nanocomplexes and SPION by the human SKOV-3 cell line (HER2 +) was investigated.Results: The SPION@CMD NPs were synthesized with spherical shape and superparamagnetic behavior. Characterization results confirmed the formation of SPION@CMD@SLTVSPWY. r2 relaxivity and r2/r1 calculations resulted in suitable values of 313 mM-1s-1 and 8.05 for SPION@CMD@SLTVSPWY, respectively. Increased uptake of targeted nanocomplexed (SPION@CMD@SLTVSPWY) compared to non-targeted NPs (SPION@CMD) was very noticeable visually, and its numerical ratio was 3.51 at a concentration of 0.075 mM. Conclusion: The targeted synthesized nanocomplex in this study has great potential as a T2 weighted probe contrast agent in MR imaging owing to its appropriate high uptake in HER2 + cells.

Synthesis of 1,4-dihydropyrano[2,3-c]pyrazole derivatives and exploring molecular and cytotoxic properties based on DFT and molecular docking studies

• Dicyclic 1,4-dihydropyran[2,3- c ]pyrazole-5-carbonitrile derivatives were synthesized as potential anticancer agents. • The cytotoxic activity was tested against PC-3, SKOV-3, HeLa and A549 cell lines. • The frontier molecular orbitals, electron affinity, ionization potential and molecular electrostatic potential (MEP) were studied. • The molecular docking was accomplished onto thymidylate synthase (TS) protein was discussed. One-pot multicomponent reaction of ethyl 3-oxobutanoate or ethyl 3-oxo-3-phenylpropanoate, hydrazine hydrate, malononitrile, various aldehydes in ethanolic piperidine solution under Microwave irradiation gave 6-amino-4-aryl/hetaryl-3-methyl/pheny-1,4-dihydropyran[2,3- c ]pyrazole-5-carbonitriles. The structures of these compounds were established on the basis of IR, 1 H NMR, 13 C NMR, 13 C NMR-APT, MS data and elemental analysis. Furthermore, the cytotoxic activity properties were evaluated against the human cancer cell lines PC-3, SKOV-3, HeLa and A549 in comparison to the positive controls Vinblastine and Doxorubicin, employing the viability assay. The obtained results confirmed that some of the tested molecules revealed strong and selective cytotoxic activities against the four cancer cell lines. Herein, frontier molecular orbitals (FMO), electron affinity (EA), ionization potential (IP) and molecular electrostatic potential (MEP) was carried out to understand the active sites and biological active nature of pyrano[2,3- c ]pyrazole synthesized compounds. A systematic study was performed then frontier molecular orbitals energies, active sites and molecular descriptors were compared with reference drug. The molecular docking was accomplished onto thymidylate synthase (TS) protein. The compounds with highest efficiency exhibited promising binding interactions and binding affinities into active site.

Arsenic compound sensitizes homologous recombination proficient ovarian cancer to PARP inhibitors

The poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors show survival benefits in ovarian cancer patients with BRCA1/2 mutation or homologous recombination (HR) deficiency, but only limited efficacy in HR-proficient ones. Another drug, arsenic trioxide (ATO) or arsenic drug (RIF), exerts antitumor effects via inducing DNA damage. Here, we investigated the combined therapeutic effects of the PARP inhibitors and the arsenic compound in HR-proficient ovarian cancer. The combined treatment of niraparib, olaparib, or fluazolepali with ATO showed a significant suppression in tumor cell viability and colony formation. The drug treatment also induced synergistic inhibition of cell proliferation and DNA damage, and acceleration of cell apoptosis in two HR-proficient ovarian cancer cell lines SKOV3 and CAOV3, either by simultaneous or sequential administration. The mechanism underlying these synergistic effects were reflected by the significantly increased ratio of cleaved-PARP/total PARP and decreased ratio of p-AKT/total AKT. Consistently, the combination of olaparib with RIF synergistically reduced the tumor growth in mouse xenograft models. In conclusion, the arsenic compound greatly sensitizes HR-proficient ovarian cancer cells to the PARP inhibitors, and our findings provide an evidence for the clinical treatment development of this combination in HR-proficient ovarian cancer patients.

Development of nimesulide analogs as a dual inhibitor targeting tubulin and HSP27 for treatment of female cancers

In drug repurposing paradigm, medicines that are already used to treat specific disease can also treat alternative ones. Recently, nimesulide was reported as an antitumor agent, beside its anti-inflammatory effect. Tubulin and heat shock protein 27 (HSP27) are attractive targets for the discovery of anticancer therapy because both are up regulated in cancer cells. In this regard, this study aims to develop nimesulide skeleton, synthesize, characterize, and biologically evaluate the new nimesulide analogs as anticancer agents through the inhibition of tubulin and HSP27 functions. The synthesized agents were characterized by 1H NMR, 13C NMR, IR, melting point and both endothermic and exothermic DSC analysis. The molecular weight was confirmed using GC–MS technique. N-(3-((2,5-dichlorobenzyl)oxy)-4-(N-methylmethylsulfonamido)phenyl)-4-iodobenzamide (agent L4) structure was confirmed using X-ray crystallographic analysis. The ligands adopt triclinic crystal with a = 8.194(14) Å, b = 10.75(3) Å, c = 14.21(2) Å and space group of P-1. Western blot analysis was used to express the presence of the dual proteins on the harvested SKOV3 and SKBR3 cell lines. The anticancer effect of nimesulide analogs was studied using MTT assay on SKOV3 and SKBR3 cell lines as a surrogate model for ovarian cancer, and breast cancer respectively. The present results indicated that the developed nimesulide analogs elicited cytotoxicity against SKOV3 and SKBR3 cell lines in turn cell cycle arrest, in the concentration range between 0.23–2.02 µM and 0.50–3.73 µM respectively. This study concluded that nimesulide analogues are potent promising agents for the repurposing of nimesulide as future chemotherapy for female cancers.

CENPA is one of the potential key genes associated with the proliferation and prognosis of ovarian cancer based on integrated bioinformatics analysis and regulated by MYBL2.

BackgroundOvarian cancer (OV) is a highly lethal disease, and the fifth leading cause of all cancer-related deaths in women. The study aimed to identify potential key genes associated with the proliferation and prognosis of OV.MethodsDifferentially expressed genes (DEGs) between ovarian cancer and normal tissues were screened by the robust rank aggregation (RRA) method. The expression of CENPA and MYBL2 were examined in SKOV3 and A2780 ovarian cancer cell lines and tumor tissues by qRT-PCR and western blot. Small RNA interference assays, plasmid overexpression assays and EdU assays were used to validate the proliferative effect of the MYBL2-CENPA axis in ovarian cancer cell lines. The ChIP assay was used to verify the direct regulation of MYBL2 on CENPA.Results133 up-regulated genes and 158 down-regulated genes were identified, and the up-regulated genes mainly enrichment in cell cycle. The three up-regulated gene with DNA separation (CENPA, CENPF and CEP55) might be tightly correlated with proliferation and prognosis of OV. Knockdown CENPA expression inhibited the proliferation of A2780 and SKOV3 cells After the knockout of MYBL2, the expression of CENPA significantly decreased. MYBL2 directly binds to the promoter region of CENPA.ConclusionsThe MYBL2-CENPA pathway plays an important role in the proliferation of ovarian cancer cells, suggesting that this pathway may be a potential target for the treatment of ovarian cancer.

Stereo‐ and Regioselective [3+3] Annulation Reaction Catalyzed by Ytterbium: Synthesis of Bicyclic 1,4‐Dihydropyridines

AbstractAn ytterbium catalyzed formal [3+3] cycloaddition of cyclic enamines and α,β‐unsaturated ketones catalyzed is reported. The reaction proceeds with a ‘head to tail’ regioselectivity through a conjugate addition of the enamine moiety followed by an amine‐carbonyl condensation. In addition the use of chiral enamines provided a high degree of stereoselectivity, driven by a possible balance between steric and π‐stacking effects. The resulting bicyclic 1,4‐dihydropyridines were evaluated as antiproliferative agents against A549 (carcinomic human alveolar basal epithelial cell) and SKOV3 (human ovarian carcinoma) human tumor cell lines. Good toxicities were found for some of the compounds against A549 and SKOV3 cell lines, with best IC50 values of 0.89 μM for A549 and 6.69 μM for SKOV3, and a very good selectivity was observed towards MRC5 (non‐malignant) cell lines.magnified image

Ultrasound‑targeted microbubble destruction‑mediated overexpression of Sirtuin 3 inhibits the progression of ovarian cancer.

Ultrasound-targeted microbubble destruction (UTMD) has recently been developed as a promising noninvasive tool for organ- and tissue-specific gene or drug delivery. The aim of the present study was to explore the role of UTMD-mediated Sirtuin 3 (SIRT3) overexpression in the malignant behaviors of human ovarian cancer (HOC) cells. Reverse transcription-quantitative PCR was performed to detect SIRT3 mRNA expression levels in normal human ovarian epithelial cells and HOC cell lines; low SIRT3 expression was found in HOC cell lines, and the SKOV3 cell line was used in the following experiments. The SIRT3-microbubble (MB) was prepared, and the effects of ultrasound-treated SIRT3-MB on biological processes of SKOV3 cells were determined. The proliferation, migration, invasion and apoptosis of SKOV3 cells were measured after SIRT3 upregulation by UTMD. Xenograft tumors in nude mice were induced to observe tumor growth in vivo. Upregulation of SIRT3 inhibited the malignant behaviors of SKOV3 cells, whereas UTMD-mediated SIRT3 upregulation further inhibited proliferation, epithelial-mesenchymal transition, invasion and migration, and induced apoptosis of SKOV3 cells, and it also inhibited tumor formation and growth in vivo. Moreover, the present study identified hypoxia inducible factor-1α (HIF-1α) as a target of SIRT3. The present study provided evidence that UTMD-mediated overexpression of SIRT3 may suppress HOC progression through the inhibition of HIF-1α.

A Multicomponent Protocol for the Synthesis of Highly Functionalized γ-Lactam Derivatives and Their Applications as Antiproliferative Agents.

An efficient synthetic methodology for the preparation of 3-amino 1,5-dihydro-2H-pyrrol-2-ones through a multicomponent reaction of amines, aldehydes, and pyruvate derivatives is reported. In addition, the densely substituted lactam substrates show in vitro cytotoxicity, inhibiting the growth of carcinoma human tumor cell lines HEK293 (human embryonic kidney), MCF7 (human breast adenocarcinoma), HTB81 (human prostate carcinoma), HeLa (human epithelioid cervix carcinoma), RKO (human colon epithelial carcinoma), SKOV3 (human ovarian carcinoma), and A549 (carcinomic human alveolar basal epithelial cell). Given the possibilities in the diversity of the substituents that offer the multicomponent synthetic methodology, an extensive structure-activity profile is presented. In addition, both enantiomers of phosphonate-derived γ-lactam have been synthesized and isolated and a study of the cytotoxic activity of the racemic substrate vs. its two enantiomers is also presented. Cell morphology analysis and flow cytometry assays indicate that the main pathway by which our compounds induce cytotoxicity is based on the activation of the intracellular apoptotic mechanism.

Stelleranoids A–M, guaiane-type sesquiterpenoids based on [5,7] bicyclic system from Stellera chamaejasme and their cytotoxic activity

Thirteen previously undescribed guaiane-type sesquiterpenoids based on [5,7] bicyclic system, stelleranoids A–M (1–13), along with six known analogues (14–20), were isolated from the roots of Stellera chamaejasme with chromatographic techniques. Their structures including absolute configurations were determined by HRESIMS and spectroscopic data, quantum chemical calculations, as well as X-ray crystallographic analysis. Cytotoxicity test in three cell lines indicated that compound 14 had relatively stronger cytotoxic effect against MKN-45, SKOV3, and Du145 cell lines with IC50 of 9.8, 17.4 and 7.3 μM, respectively; compounds 3 and 8 displayed moderate cytotoxic effect against MKN-45 and Du145 cell lines with IC50 ranged from 14.5 to 18.8 μM, comparable to those of the positive control. As determined by fluorescent microscopy and flow cytometry in Du145 cell line, compound 14 could promote cell apoptosis and cause cell cycle arrest at the G0/G1 phase, leading to the inhibition of cell proliferation. Further Western blot analysis revealed that this inhibitory effect was accompanied by upregulating pro-apoptosis proteins cleaved-PARP, cleaved-Caspase-9 and tumor suppressor protein p53 while downregulating anti-apoptotic protein Bcl-2 in 14-treated Du145 cells.

Connections between prolactin and ovarian cancer.

Ovarian cancer (OC) is characterized by a high morbidity and mortality, highlighting a great need for a better understanding of biological mechanisms that affect OC progression and improving its early detection methods. This study investigates effects of prolactin (PRL) on ovarian cancer cells, analyzes PRL receptors (PRLR) in tissue micro arrays and relates PRLR expression to survival of ovarian cancer. A database, composed of transcript profiles from OC, was searched for PRLR expression and results were put in relation to survival. Expression of PRLR in OC tissue sections and OC cell lines SKOV3, OV2008 and OVSAHO was assessed using immunohistochemistry, western blots and quantitative real-time PCR. The biological function of PRLR was evaluated by proliferation, colony formation and wound healing assays. Levels of PRLR mRNA are related to survival; in epithelial OC a high PRLR mRNA expression is related to a shorter survival. Analysis of a tissue micro array consisting of 84 OC showed that 72% were positive for PRLR immuno-staining. PRLR staining tended to be higher in OC of high grade tumors compared to lower grades. PRLR mRNA and protein can further be detected in OC cell lines. Moreover, in vitro treatment with PRL significantly activated the JAK/STAT pathway. PRLR expression is associated with OC survivals. PRL and its receptor may play an onco-modulatory role and promote tumor aggressiveness in OC. Alternatively, increased PRLR levels may form a base for the development of PRLR antagonist or PRLR antagonist-drug conjugate to increase selective uptake of anti-cancer drugs.

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.

Histone deacetylase inhibitor induced pVHL-independent degradation of HIF-1α and hierarchical quality control of pVHL via chaperone system.

The mortality rate of ovarian cancer is increasing and the role of hypoxia inducible factor-1α (HIF-1α) in tumor progression has been confirmed. von Hippel-Lindau tumor suppressor protein (pVHL) binds HIF-1α and mediates proteasome degradation of HIF-1α. Besides, histone deacetylase inhibitor (HDACi) mitigates tumor growth via targeting HIF-1α, whereas underlying mechanism still requires investigation. In this research, we exposed ovarian cancer cell lines OV-90 and SKOV-3 to escalating concentrations of HDACi LBH589. As a result, cell viability was significantly suppressed and expression of HIF-1α was remarkably reduced along with decreased levels of signal molecules, including phosphoinositide 3-kinase (PI3K) and glycogen synthase kinase 3β (GSK3β) (P = 0.000). Interestingly, pVHL was expressed in a notably declining tendency (P = 0.000). Chaperone heat shock protein-70 (HSP70) was expressed in an ascending manner, whereas expression of chaperonin TCP-1α was reduced clearly (P = 0.000). Besides, co-inhibition of pVHL plus HDAC did not contribute to a remarkable difference in HIF-1α expression as compared with single HDAC inhibition. Furthermore, both cell lines were transfected with plasmids of VHL plus VHL binding protein-1 (VBP-1). Consequently, the expression of HIF-1α as well as lactate dehydrogenase-A (LDHA) was remarkably decreased (P = 0.000). These findings indicate HDACi may repress expression of HIF-1α via inhibiting PI3K and GSK3β and promote degradation of HIF-1α via HSP70, independent of pVHL. Additionally, a sophisticated network of HDAC and chaperones may involve in pVHL quality control.

IFN-γ inhibits ovarian cancer progression via SOCS1/JAK/STAT signaling pathway.

Ovarian cancer (OC) is a common malignancy, and IFN-γ, a multifunctional cytokine, is unveiled to impede the multiplication and enhance apoptosis in diverse tumor cells in previous research. Nonetheless, its function and mechanism in OC are blurred. OC cell lines SKOV3 and OVCAR3 were dealt with different concentrations (0-40ng/ml) of IFN-γ. CCK-8 experiment was utilized to examine cell multiplication; Flow cytometry was executed to detect apoptosis and cell cycle; Wound healing assay was utilized to detect cell migration; and Transwell experiment was implemented to examine cell invasion. qRT-PCR analysis was applied to detect STAT5, STAT3, JAK2 and JAK3 mRNA expression in OC cell lines. Western blot experiment was applied to detect the protein and phosphorylation levels of SOCS1, STAT5 and STAT3. IFN-γ suppressed OC cell multiplication in a concentration-dependent manner. Relative to the control group, IFN-γ restrained OC cell migration, invasion, enhanced apoptosis and prevented cell transformation from G0/G1 to S phase. Further analysis revealed that IFN-γ up-modulated SOCS1 expression and impeded STAT5 and STAT3 protein phosphorylation levels, and knockdown of SOCS1 partially counteracted the inhibitory effect of IFN-γ on STAT5 and STAT3 protein phosphorylation levels. IFN-γ represses OC progression by facilitating SOCS1 to suppress STAT3 and STAT5 protein phosphorylation.

CD70 antibody-drug conjugate: A potential novel therapeutic agent for ovarian cancer.

This study aimed to investigate the cytotoxicity of a cluster of differentiation 70 antibody‐drug conjugate (CD70‐ADC) against ovarian cancer in in vitro and in vivo xenograft models. CD70 expression was assessed in clinical samples by immunohistochemical analysis. Western blotting and fluorescence‐activated cell sorting analyses were used to determine CD70 expression in the ovarian cancer cell lines A2780 and SKOV3, and in the cisplatin‐resistant ovarian cancer cell lines A2780cisR and SKOV3cisR. CD70 expression after cisplatin exposure was determined in A2780 cells transfected with mock‐ or nuclear factor (NF)‐κB‐p65‐small interfering RNA. We developed an ADC with an anti‐CD70 monoclonal antibody linked to monomethyl auristatin F and investigated its cytotoxic effect. We examined 63 ovarian cancer clinical samples; 43 (68.3%) of them expressed CD70. Among patients with advanced stage disease (n = 50), those who received neoadjuvant chemotherapy were more likely to exhibit high CD70 expression compared to those who did not (55.6% [15/27] vs 17.4% [4/23], P < .01). CD70 expression was confirmed in A2780cisR, SKOV3, and SKOV3cisR cells. Notably, CD70 expression was induced after cisplatin treatment in A2780 mock cells but not in A2780‐NF‐κB‐p65‐silenced cells. CD70‐ADC was cytotoxic to A2780cisR, SKOV3, and SKOV3cisR cells, with IC50 values ranging from 0.104 to 0.341 nmol/L. In A2780cisR and SKOV3cisR xenograft models, tumor growth in CD70‐ADC treated mice was significantly inhibited compared to that in the control‐ADC treated mice (A2780cisR: 32.0 vs 1639.0 mm3, P < .01; SKOV3cisR: 232.2 vs 584.9 mm3, P < .01). Platinum treatment induced CD70 expression in ovarian cancer cells. CD70‐ADC may have potential therapeutic implications in the treatment of CD70 expressing ovarian cancer.