OVCAR-3 Cells Research Articles

Synthesis and biological activity of bisindole derivatives as novel MARK4 inhibitors

Bisindole alkaloids are well-known bioactive natural products presenting numerous pharmacological properties. Following the indolocarbazole biosynthetic pathway a series of bisindole derivatives were synthesized and their activity against MARK4 kinase was investigated. The binding of bisindoles on MARK4 was measured with both fluorescence quenching and ITC. Further, enzyme inhibition assays revealed that the tested compounds are MARK4 inhibitors with IC50 values in the low micromolar range. The inhibitory activity of bisindoles 20 and 33 was tested against four additional kinases. The data showed that 20 and 33 have a relatively low selectivity against MARK4 as they also moderately inhibit other kinases such as PDK3. Most of the synthesized compounds were subjected to molecular docking studies to rationalise the molecular interactions within the catalytic site of MARK4. The antiproliferative activity of derivatives 18, 26, 33 and 20 was evaluated against A549, MCF7 and OVCAR-3 cancer cells. Among the tested compounds, 20 displayed the best cytotoxic activity against OVCAR-3 ​cells with GI50 ​= ​7 ​± ​0.5 μΜ, TGI ​= ​10 ​± ​0.8 μΜ and IC50 ​= ​20 ​± ​1.2 μΜ and A549 cancer cells with GI50 ​= ​4 ​± ​0.52 μΜ, TGI ​= ​12.5 ​± ​0.8 μΜ and IC50 32.5 ​± ​2.4 ​μM. In addition, compounds 18 and 26 induce apoptosis in A549 ​cells and deviated the cells from early to late apoptotic events. Overall, the new bisindole derivatives possess significant antiproliferative properties, yet a non-selective cytotoxic effect was observed on normal MRC5 cells. Our observations support the suitability of bisindole scaffold for the design of potent MARK4 inhibitors, but to improve the drugability further optimization and structural modifications are necessary.

Anti-Tumoral Effect of Chemerin on Ovarian Cancer Cell Lines Mediated by Activation of Interferon Alpha Response

Simple SummaryChemerin is a multifunctional protein with an important role in the immune system. Recent evidence showed that chemerin also regulates the development of cancer. Ovarian cancer is a common type of tumor in women. In this study, we observed that chemerin decreases the growth of ovarian cancer cell lines in vitro when cultivated in standard cell culture or in globular multicellular aggregates. When we examined the mechanisms involved in this process, we found that treatment of ovarian cancer cells with chemerin led to the activation of genes that are known to mediate the effects of interferon alpha (IFNα). The main effect of IFNα is to defend body cells against viral infections, but it is also able to defeat cancer cells. We observed that this activation of IFNα response by chemerin resulted from the increased production of IFNα protein in ovarian cancer cells, which then reduced cancer cells numbers. However, it remains to be investigated how exactly chemerin might be able to activate interferon alpha and its anti-tumoral actions.The pleiotropic adipokine chemerin affects tumor growth primarily as anti-tumoral chemoattractant inducing immunocyte recruitment. However, little is known about its effect on ovarian adenocarcinoma. In this study, we examined chemerin actions on ovarian cancer cell lines in vitro and intended to elucidate involved cell signaling mechanisms. Employing three ovarian cancer cell lines, we observed differentially pronounced effects of this adipokine. Treatment with chemerin (huChem-157) significantly reduced OVCAR-3 cell numbers (by 40.8% on day 6) and decreased the colony and spheroid growth of these cells by half. The spheroid size of SK-OV-3 ovarian cancer cells was also significantly reduced upon treatment. Transcriptome analyses of chemerin-treated cells revealed the most notably induced genes to be interferon alpha (IFNα)-response genes like IFI27, OAS1 and IFIT1 and their upstream regulator IRF9 in all cell lines tested. Finally, we found this adipokine to elevate IFNα levels about fourfold in culture medium of the employed cell lines. In conclusion, our data for the first time demonstrate IFNα as a mediator of chemerin action in vitro. The observed anti-tumoral effect of chemerin on ovarian cancer cells in vitro was mediated by the notable activation of IFNα response genes, resulting from the chemerin-triggered increase of secreted levels of this cytokine.

Involvement of Bid in the crosstalk between ferroptotic agent-induced ER stress and TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces death receptor-mediated extrinsic apoptosis, specifically in cancer cells, and Bid (BH3-interacting domain death agonist) plays an important role in TRAIL-induced apoptosis. Ferroptosis is a newly defined form of regulated cell death known to be distinct from other forms of cell death. However, our previous studies have shown that ferroptosis shares common pathways with other types of programmed cell death such as apoptosis. In this study, we investigated the role of Bid in the crosstalk between the ferroptotic agent-induced endoplasmic reticulum (ER) stress response and TRAIL-induced apoptosis. When human colorectal carcinoma HCT116 cells were treated with the ferroptosis-inducing agents artesunate and erastin in combination with TRAIL, TRAIL-induced activation of caspase-8 was enhanced, and subsequently, the truncation of Bid was increased. Similar results were observed when ovarian adenocarcinoma OVCAR-3 cells were treated with the ferroptotic agents in combination with TRAIL. Results from studies with Bid mutants reveal that the truncation of Bid and the presence of intact BH3 domains are critical for synergistic apoptosis. Nonfunctional Bid mutants were not able to activate the mitochondria-dependent apoptosis pathway, which is required for the conversion of p19 to p17, the active form of caspase-3. These results indicate that Bid plays a critical role in the crosstalk between the ferroptotic agent-induced ER stress response and TRAIL-induced apoptosis.

Amlexanox-modified platinum(IV) complex triggers apoptotic and autophagic bimodal death of cancer cells

Platinum(IV) prodrugs c,c,t-[PtCl2(NH3)2(OH)(amlexanox)] (MAP) and c,c,t-[PtCl2(NH3)2(amlexanox)2] (DAP) were synthesized by reacting amlexanox with oxoplatin and characterized by NMR, HR-MS, HPLC, and elemental analysis. The complexes could be reduced to platinum(II) species and amlexanox to exert antitumor activity. Generally, MAP was more potent than DAP and cisplatin towards various human cancer cell lines; particularly, it was active in cisplatin-resistant Caov-3 ovarian cancer and A549/DDP lung cancer cells. MAP induced serious damage to DNA, remarkable change in mitochondrial morphology, decrease in mitochondrial membrane potential, release of cytochrome c from mitochondria, and up-regulation of pro-apoptotic protein Bax in Caov-3 cells, thereby leading to evident apoptosis. Meanwhile, MAP markedly promoted the autophagic flux, including affecting the expression of microtubule-associated protein light chain 3 (LC3) and autophagy adaptor protein p62 in Caov-3 cells, with an increase in the ratio of LC3-II/LC3-I and a decrease in p62, thus trigging the occurrence of autophagy. The MAP-induced bimodal cell death mode is uncommon for platinum complexes, which presents a new possibility to invent anticancer drugs with unique mechanism of action.

Overexpression of NCAPG in ovarian cancer is associated with ovarian cancer proliferation and apoptosis via p38 MAPK signaling pathway

BackgroundNon-SMC condensin I complex subunit G (NCAPG), a member of the subunit of condensin complex, is significantly overexpressed in various cancers and involved in the pathogenesis of cancers. However, the roles of NCAPG in ovarian cancer remain unclear.MethodsThe mRNA expression, overall survival, and disease-free survival of NCAPG in ovarian cancer were analyzed by GEPIA and KM plotter database, and the expression levels of NCAPG in OC tissues and cell lines were determined by qPCR and immunohistochemistry analysis. shRNA targeting NCAPG gene (sh-NCAPG) was utilized to knock down NCAPG expression in OVCAR3 and SKOV3 cells. Subsequently, CCK-8 assay, colony formation assay, transwell invasion assay and flow cytometric analysis were performed to detect the effect of NCAPG on OC cell proliferation, apoptosis, and invasion. Finally, western blot assays were performed to detect the mechanism of NCAPG in ovarian cancer.ResultsAnalysis using GEPIA and KM plotter database showed NCAPG was upregulated in ovarian cancer and negatively associated with the survival of OC patients. qPCR and immunohistochemistry analysis confirmed it was highly expressed in both ovarian cancer tissues and cells. The silencing of NCAPG inhibited OC cell proliferation and invasion, and induced cell apoptosis. Additionally, flow cytometric analysis revealed that NCAPG knockdown arrested the cell cycle at G2 and S phases. Furthermore, we also found that downregulation of NCAPG could suppress OC cell proliferation and invasion via activating the p38 MAPK signaling pathway.ConclusionOur results suggest that NCAPG exhibits an important role in the development and progression of ovarian cancer and implicates NCAPG as a potential therapeutic target in ovarian cancer.

Recognition of mutant forms of the sodium-dependent phosphate transporter NaPi2b by ­monoclonal antibodies in ovarian cancer cells

Background. The sodium-dependent phosphate transporter NaPi2b is expressed in a number of tumors and serves as a target for monoclonal antibody therapy. The NaPi2b transporter has a large extracellular domain containing 4 cysteines and an MX35 epitope recognized by the corresponding monoclonal antibodies.
 Aim. To study the recognition of the MX35 epitope in cysteine mutants C303 and C350 of the sodium-dependent phosphate transporter NaPi2b by monoclonal antibodies in ovarian cancer cells.
 Material and methods. Ovarian cancer cells of the OVCAR-8 line were transfected with plasmids encoding mutant forms of the NaPi2b transporter. The recognition of these forms by monoclonal antibodies was studied by Western blot and flow cytometry. Statistical analysis of flow cytometry data was performed using Pearsons Chi-squared test with Yates correction.
 Results. Western blot analysis of lysates of OVCAR-8 cells expressing the wild-type NaPi2b transporter demonstrated the presence of a specific signal only in samples without the addition of dithiothreitol, while in all samples with NaPi2b cysteine mutants, even without the addition of dithiothreitol, no specific signal from monoclonal antibodies was detected. The flow cytometry results showed that proportion of OVCAR-8 cells with positive staining of the MX35 epitope with antibodies is less when expressing the NaPi2b mutants compared to the wild type.
 Conclusion. Cysteines C303 and C350 are involved in disulfide bonds formation, which is of key importance for the formation of the conformation of the extracellular domain of the sodium-dependent phosphate transporter NaPi2b, which ensures the MX35 epitope recognition by monoclonal antibodies.

Odd-skipped related 1 plays a tumor suppressor role in ovarian cancer via promoting follistatin-like protein 1 transcription.

Zinc-finger transcription factor odd-skipped related 1 (OSR1) is involved in the progression of certain types of cancers, via regulating the transcription of downstream genes. However, the function of OSR1 in ovarian cancer (OC) progression remains unclear. The present study aimed to explore the OSR1 expression pattern in OC tissues and cell lines. Functional assays were performed to explore the regulatory effects of OSR1 on OC cell growth, migration and invasion in vitro and in vivo. Results of the present study demonstrated that OSR1 was significantly downregulated in OC tissues compared with healthy ovarian tissues (P < 0.01). Moreover, SKOV-3 and OVCAR-3 cells with low OSR1 expression were used for functional studies, and results demonstrated that OSR1 overexpression suppressed cell growth by inhibiting cell cycle progression and inducing cell apoptosis in vitro. OC cells with higher OSR1 expression levels exhibited reduced levels of migration and invasion, when compared with the corresponding control. In addition, OSR1 expression in xenografts models resulted in diminished tumor volume and suppressed tumorigenesis. OSR1 enhanced follistatin-like protein 1 (FSTL1) expression at the transcriptional level through directly binding to the promoter of FSTL1, which was commonly reported to exert a tumor suppressor role in OC progression. Moreover, FSTL1 knockdown reversed the action of OSR1 overexpression in OC progression, including cell viability, migration, invasion, and apoptosis. In conclusion, these results indicated that OSR1 may function as a tumor suppressor through augmenting FSTL1 transcription in OC progression, suggesting that the OSR1/ FSTL1 axis may exhibit potential as a therapeutic target for OC therapy.

WISP1 induces ovarian cancer via the IGF1/αvβ3/Wnt axis

BackgroundThis study intended to clarify the mechanisms by which WISP1-mediated IGF1/αvβ3/Wnt axis might affect the progression of ovarian cancer.MethodsBioinformatics analysis was implemented for pinpointing expression of IGF1 and WISP1 which was verified through expression determination in clinical tissue samples and cells. Next, gain- or loss-of-function experimentations were implemented for testing CAOV4 and SKOV3 cell biological processes. The interaction between WISP1 and IGF1 was verified by co-immunoprecipitation and the molecular mechanism was analyzed. Finally, ovarian cancer nude mouse models were prepared to unveil the in vivo effects of WISP1/IGF1.ResultsIGF1 and WISP1 expression was elevated in ovarian cancer tissues and cells, which shared correlation with poor prognosis of ovarian cancer sufferers. Elevated IGF1 induced malignant properties of ovarian cancer cells through activation of PI3K-Akt and Wnt signaling pathway. WISP1 was positively correlated with IGF1. WISP1 could enhance the interaction between IGF1 and αvβ3 to induce epithelial-mesenchymal transition. In vivo experiments also confirmed that upregulated WISP1/IGF1 induced tumorigenesis and metastasis of ovarian cancer cells.ConclusionIn conclusion, WISP1 can facilitate ovarian cancer by activating Wnt via the interaction between IGF1 and αvβ3.

Homoisoflavonoids from the bulbs of Bellevalia longipes and an assessment of their potential cytotoxic activity

Seven undescribed homoisoflavonoids were identified from the bulbs of Bellevalia longipes Post (Asparagaceae) as well as thirteen known and one natural homoisoflavonoid that had been reported as a synthetic product previously. A general approach for recognizing homoisoflavonoids via NMR spectroscopy data were presented. The undescribed compounds were: 8-dehydroxy-5-O-demethyl-6-hydroxyscillapersicone, 6-methoxyscillapersicone, 5-O-demethyl-6-methoxyscillapersicone, 8-O-methylscillapersicone, 4′-O-methylscillapersicone, 4′,8-O,O-dimethylscillapersicone, 3′-O-methylscillapersicone, and 3-hydroxy-desmethylophiopogonanone A. Structures were determined based on analysis of HRMS and NMR data, while absolute configurations were assigned using ECD spectroscopy. Human cancer cell lines were used to assess the cytotoxic activities of the isolated compounds, where 3-dehydroxy-3′-hydroxyeucomol showed IC50 values of 0.62 μM, 5.36 μM, and 2.52 μM, when tested against MDA-MB-435 (melanoma), MDA-MB-231 (breast), and OVCAR3 (ovarian) cells, respectively.

RAD54B mutations enhance the sensitivity of ovarian cancer cells to poly(ADP-ribose) polymerase (PARP) inhibitors

Synthetic lethal targeting of homologous recombination (HR)–deficient ovarian cancers (OvCas) with poly(ADP-ribose) polymerase inhibitors (PARPis) has attracted considerable attention. Olaparib was the first PARPi approved by the Food and Drug Administration, offering significant clinical benefits in BRCA1/2-deficient OvCas. However, only approximately 20% of OvCa patients harbor BRCA1/2 mutations. Given the shared roles that BRCA1/2 have with other HR regulators, alterations in HR genes may also contribute to “BRCAness profiles” in OvCas. RAD54B has been considered a key player in HR repair, although its roles and therapeutic potential in cancers need further investigation. Here, we identified 22 frequently mutated HR genes by whole-exome sequencing of OvCa tissues from 82 patients. To our surprise, 7.3% of patients were found to harbor mutations of RAD54B, the third-highest mutated gene among patients. We determined that RAD54B-mutated tumor tissues harbored more DNA double-strand breaks than normal tissues. Additionally, we found that RAD54B knockdown inhibited HR repair, enhanced sensitivities of OvCa cells with increased DNA double-strand breaks to olaparib, and induced apoptosis. Enhanced inhibitory effects of olaparib on the growth of ES2 xenograft tumors were further demonstrated by RAD54B knockdown. Finally, we show that restoration with wildtype RAD54B rather than RAD54BN593S and RAD54BH219Y, identified in patients, abolished the effects of RAD54B knockdown, indicating these RAD54B mutants probably malfunctioned in HR repair. Our investigations may offer insight into the contributions of RAD54B mutations to synthetic lethality with olaparib treatment in OvCas, enrich the gene list for “HR deficiency scoring,” and expand the applications of PARPis.

Molecular Mechanisms of Cytotoxicity of NCX4040, the Non-Steroidal Anti-Inflammatory NO-Donor, in Human Ovarian Cancer Cells.

NCX4040, the non-steroidal anti-inflammatory-NO donor, is cytotoxic to several human tumors, including ovarian tumor cells. We have found that NCX4040 is also cytotoxic against both OVCAR-8 and its adriamycin resistant (NCI/ADR-RES) tumor cell lines. Here, we have examined mechanism(s) for the cytotoxicity of NCX4040 in OVCAR-8 and NCI/ADR-RES cell lines. We found that NCX4040 induced significant apoptosis in both cell lines. Furthermore, NCX4040 treatment caused significant depletion of cellular glutathione, causing oxidative stress due to the formation of reactive oxygen/nitrogen species (ROS/RNS). Significantly more ROS/RNS were detected in OVCAR-8 cells than in NCI/ADR-RES cells which may have resulted from increased activities of SOD, glutathione peroxidase and transferases expressed in NCI/ADR-RES cells. NCX4040 treatment resulted in the formation of double-strand DNA breaks in both cells; however, more of these DNA breaks were detected in OVCAR-8 cells. RT-PCR studies indicated that NCX4040-induced DNA damage was not repaired as efficiently in NCI/ADR-RES cells as in OVCAR-8 cells which may lead to a differential cell death. Pretreatment of OVCAR-8 cells with N-acetylcysteine (NAC) significantly decreased cytotoxicity of NCX4040 in OVCAR-8 cells; however, NAC had no effects on NCX4040 cytotoxicity in NCI/ADR-RES cells. In contrast, FeTPPS, a peroxynitrite scavenger, completely blocked NCX4040-induced cell death in both cells, suggesting that NCX4040-induced cell death could be mediated by peroxynitrite formed from NCX4040 following cellular metabolism.

Intra-peritoneal CAR-T cell therapy shows promising results in a murine model of epithelial ovarian cancer (313)

<b>Objectives:</b> Epithelial ovarian cancer is often diagnosed at an advanced stage due to intraperitoneal spread. Modern treatment strategies have only modestly improved the survival rates of this disease. Chimeric antigen receptor (CAR) T cells enable T cells to directly bind tumor-associated antigens in an MHC-independent manner inducing tumor rejection. A major barrier is the lack of true tumor-specific antigens followed by risk of "on-target off-tumor" toxicity. Innovative approaches are therefore needed to increase the specificity of CAR-modified T cells exclusively against tumors. The aim of this study was to assess the efficacy and safety of intraperitoneal versus intravenous CAR T cell therapy. Our working hypothesis was that intraperitoneal CAR T cell therapy will demonstrate higher cytotoxic activity towards a tumor tissue while sparing non-tumor tissues. <b>Methods:</b> We have constructed CARs targeting <i>HER2</i> (ErbB2) that are overexpressed in ovarian cancers. CARs were expressed in human lymphocytes, and their functionality toward human ovarian cancer cell lines was evaluated <i>in-vitro</i> and in a murine model. <b>Results:</b> We have found that the anti-ErbB2 CARs secreted IFN-g (600-1800pg/ml) after stimulation with OVCAR8, SKOV3, or NAR cells compared to untransduced lymphocytes CAR T cells (50pg/ml). Treatment with an intraperitoneal injection of anti-ErbB2 CAR T cells leads to remission of the tumor and increased survival compared to the intravenous route. Intraperitoneal treatment of anti-ErbB2 CARs was less toxic to non-tumor tissues compared to the intravenous treatment. <b>Conclusions:</b> Our results demonstrate that the intra-peritoneal approach of CAR T cells offers a safe strategy with a clinical potential for the treatment of ovarian cancer.

Efficacy of combining tumor treating fields (TTFields) with a PARP inhibitor in ovarian cell lines (300.5)

Objectives: PARP inhibition demonstrates clinical effectiveness in patients with solid tumors and deleterious BRCA mutations. The use of PARP inhibitors is specifically relevant in ovarian cancer, as one of three women with this malignancy harbor BRCA mutations. Tumor Treating Fields (TTFields) are alternating electric fields with antimitotic effects on cancerous cells, which are approved to treat patients with glioblastoma and malignant pleural mesothelioma. TTFields were shown to induce a state of BRCAness in several cancerous cell lines. The objective of this study was to explore the potential of combining TTFields with PARP inhibition for the treatment of ovarian cancer based on in vitro work. Methods: Ovarian carcinoma cells, A2780 (BRCA1/2 wild type) and OVCAR3 (BRCA1 wild type, BRCA2 mutated), were treated for 72 h with TTFields (at an intensity of 1.2 V/cm RMS and frequency of 200 kHz) using the in vitro system. The effectiveness of TTFields in combination with the PARP inhibitor niraparib was examined by measuring cell counts, clonogenic potential, overall effect (multiplicity product of cytotoxicity and clonogenicity), and apoptosis induction. Results: The application of TTFields to A2780 or OVCAR3 cells resulted in about 50% reduction in cell counts. Niraparib displayed a dose-dependent cytotoxic effect on both cell lines. Co-application of niraparib and TTFields resulted in enhanced cytotoxicity compared to the effect of niraparib or TTFields alone. TTFields also elevated the effects of niraparib on the colony-forming ability of the cells, the overall effect, and apoptosis. Conclusions: These results suggest a potential benefit for concomitant treatment of TTFields with niraparib in ovarian cancer. Additional experiments will examine the effect on the BRCA-FANC pathway in ovarian cancer. Objectives: PARP inhibition demonstrates clinical effectiveness in patients with solid tumors and deleterious BRCA mutations. The use of PARP inhibitors is specifically relevant in ovarian cancer, as one of three women with this malignancy harbor BRCA mutations. Tumor Treating Fields (TTFields) are alternating electric fields with antimitotic effects on cancerous cells, which are approved to treat patients with glioblastoma and malignant pleural mesothelioma. TTFields were shown to induce a state of BRCAness in several cancerous cell lines. The objective of this study was to explore the potential of combining TTFields with PARP inhibition for the treatment of ovarian cancer based on in vitro work. Methods: Ovarian carcinoma cells, A2780 (BRCA1/2 wild type) and OVCAR3 (BRCA1 wild type, BRCA2 mutated), were treated for 72 h with TTFields (at an intensity of 1.2 V/cm RMS and frequency of 200 kHz) using the in vitro system. The effectiveness of TTFields in combination with the PARP inhibitor niraparib was examined by measuring cell counts, clonogenic potential, overall effect (multiplicity product of cytotoxicity and clonogenicity), and apoptosis induction. Results: The application of TTFields to A2780 or OVCAR3 cells resulted in about 50% reduction in cell counts. Niraparib displayed a dose-dependent cytotoxic effect on both cell lines. Co-application of niraparib and TTFields resulted in enhanced cytotoxicity compared to the effect of niraparib or TTFields alone. TTFields also elevated the effects of niraparib on the colony-forming ability of the cells, the overall effect, and apoptosis. Conclusions: These results suggest a potential benefit for concomitant treatment of TTFields with niraparib in ovarian cancer. Additional experiments will examine the effect on the BRCA-FANC pathway in ovarian cancer.

Tuning the Biological Activity of Camphorimine Complexes through Metal Selection.

The cytotoxic activity of four sets of camphorimine complexes based on the Cu(I), Cu(II), Ag(I), and Au(I) metal sites were assessed against the cisplatin-sensitive A2780 and OVCAR3 ovarian cancer cells. The results showed that the gold complexes were ca. one order of magnitude more active than the silver complexes, which in turn were ca. one order of magnitude more active than the copper complexes. An important finding was that the cytotoxic activity of the Ag(I) and Au(I) camphorimine complexes was higher than that of cisplatin. Another relevant aspect was that the camphorimine complexes did not interact significantly with DNA, in contrast with cisplatin. The cytotoxic activity of the camphorimine complexes displayed a direct relationship with the cellular uptake by OVCAR3 cells, as ascertained by PIXE (particle-induced X-ray emission). The levels of ROS (reactive oxygen species) formation exhibited an inverse relationship with the reduction potentials for the complexes with the same metal, as assessed by cyclic voltammetry. In order to gain insight into the toxicity of the complexes, their cytotoxicity toward nontumoral cells (HDF and V79 fibroblasts) was evaluated. The in vivo cytotoxicity of complex 5 using the nematode Caenorhabditis elegans was also assessed. The silver camphorimine complexes displayed the highest selectivity coefficients (activity vs. toxicity).

CBX8 Together with SET Facilitates Ovarian Carcinoma Growth and Metastasis by Suppressing the Transcription of SUSD2.

Our study uncovers a novel mechanism CBX8 explores to execute gene repression, and provides new therapeutic targets for ovarian carcinoma.

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

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

Structure-Based Identification and Biological Characterization of New NAPRT Inhibitors.

NAPRT, the rate-limiting enzyme of the Preiss–Handler NAD biosynthetic pathway, has emerged as a key biomarker for the clinical success of NAMPT inhibitors in cancer treatment. Previous studies found that high protein levels of NAPRT conferred resistance to NAMPT inhibition in several tumor types whereas the simultaneous blockade of NAMPT and NAPRT results in marked anti-tumor effects. While research has mainly focused on NAMPT inhibitors, the few available NAPRT inhibitors (NAPRTi) have a low affinity for the enzyme and have been scarcely characterized. In this work, a collection of diverse compounds was screened in silico against the NAPRT structure, and the selected hits were tested through cell-based assays in the NAPRT-proficient OVCAR-5 ovarian cell line and on the recombinant hNAPRT. We found different chemotypes that efficiently inhibit the enzyme in the micromolar range concentration and for which direct engagement with the target was verified by differential scanning fluorimetry. Of note, the therapeutic potential of these compounds was evidenced by a synergistic interaction between the NAMPT inhibitor FK866 and the new NAPRTi in terms of decreasing OVCAR-5 intracellular NAD levels and cell viability. For example, compound IM29 can potentiate the effect of FK866 of more than two-fold in reducing intracellular NAD levels. These results pave the way for the development of a new generation of human NAPRTi with anticancer activity.

Novel LncRNA ZFHX4-AS1 as a Potential Prognostic Biomarker That Affects the Immune Microenvironment in Ovarian Cancer.

BackgroundOvarian cancer (OvCa) is a malignant disease of the female reproductive system with a high mortality rate. LncRNA has been confirmed to play a crucial role in the development and progression of various cancer types. Novel lncRNA ZFHX4-AS1 has been reported in several cancers, albeit its functional mechanisms in OvCa remain unclear.MethodsWith reference to the public databases and based on integrating bioinformatics analyses, we explored the expression of ZFHX4-AS1 and its roles in the prognosis of OvCa. We employed the Kaplan-Meier curves to investigate the outcome of patients with different ZFHX4-AS1 expressions. Furthermore, its biological function and the related hallmark pathways were assessed through Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, and Gene-set enrichment analysis (GSEA). We explored the correlation between lncRNA ZFHX4-AS1 and tumor-infiltrating immune cells through CIBERSORT. The immune checkpoints associated with lncRNA ZFHX4-AS1 and its related genes were investigated. The effect of lncRNA ZFHX4-AS1 on proliferation, invasion and migration of OvCa cells was verified through Cell Counting Kit (CCK)-8, colony formation, wound healing and transwell assays.ResultsThe expression of lncRNA ZFHX4-AS1 was upregulated in OvCa relative to that in normal tissues. Increased lncRNA ZFHX4-AS1 expression was associated with poor overall survival and progression-free survival in OvCa. The GO and KEGG pathway analyses revealed the role of lncRNA ZFHX4-AS1 in cell metabolism, protein synthesis, cell proliferation, and cell cycle. GSEA indicated the hallmark gene sets that were significantly enriched in the high and low expression groups. The CIBERSORT database revealed M2 macrophages, memory B-cells, naïve B cells, and activated NK cells were affected by lncRNA ZFHX4-AS1 expression (all P < 0.05). The expression of lncRNA ZFHX4-AS1 and its related differential genes MRPS11, NSA2, and MRPL13 were significantly correlated with the immune checkpoints. Knockdown of lncRNA ZFHX4-AS1 could inhibit the proliferation, invasion and migration of OvCa cells.ConclusionsThe results suggested that lncRNA ZFHX4-AS1 is a novel prognostic biomarker associated with cell proliferation, metabolism, infiltration, and distribution of tumor-infiltrating immune cells in OvCa, indicating that lncRNA ZFHX4-AS1 can be used as a potential therapeutic target for OvCa in the future.

Effects of Valproic Acid with Different Drugs in Ovarian Cancer Cell Lines: An in vitro Study

Ovarian cancer is one of the most lethal malignancies, ranked seventh among all cancers globally. In recent trends, there has been an increased use of natural compounds combined with conventional drugs in clinical trials to combat chemotherapeutic resistance. Valproic acid, a short-chain fatty acid, Resveratrol, Silymarin, and Cyclophosphamide are some of the compounds widely studied for various cancer treatments. Hence, an in vitro attempt at the combinatorial effect of drugs was investigated in human ovarian adenocarcinoma cells OVCAR-3 and SKOV-3. Cytotoxicity, apoptosis, and antioxidant scavenging activity were studied by MTT, AO/EB, and DPPH assay, respectively. In addition, analysis of quantitative residual protein of treated cell culture supernatants was evaluated in OVCAR-3 and SKOV-3. The combination of valproic acid with resveratrol and silymarin showed effective IC50 and apoptosis when compared with cyclophosphamide. The antioxidant activity of valproic acid in combination with resveratrol and silymarin was 88% and 81%, respectively, at 50μg/ml. The study highlights the effective treatment strategy of combinatorial drugs for ovarian cancer. However, the limitations extend towards a molecular approach in the future for further validation with various other in vitro methods and preclinical studies.

ERCC5 abnormal expression influences the biological behaviour of ovarian cancer by Wnt pathway

Nucleotide excision repair (NER) is the most important repair pathway of DNA, and a variety of cancers are closely related to the gene abnormal expression and the deletion or downregulation of gene function in NER pathway. In this study, we investigated the differential expression of ERCC5 in different ovarian cancer cells and its potential mechanism. By detecting the expression of ovarian in both SKOV3 and Caov4 cell lines, we found that the expression of ERCC5 in SKOV3 was significantly higher than that in Caov4. We further constructed the SKOV3 cell line with low ERCC5 expression by RNA interference and Caov4 cell line with high ERCC5 expression by transient transfection, then comparing the effect of ERCC5 abnormal expression on the biological behaviour of ovarian cancer cells. The results showed that high expression of ERCC5 could significantly promote proliferation and migration of ovarian cancer but inhibit apoptosis. However, low expression of ERCC5 could significantly promote apoptosis but inhibited proliferation and migration. We further observed the effect of ERCC5 abnormal expression on potential regulation pathway by Western blot and noticed that ERCC5 abnormal expression could activate the Wnt pathway. Overall, the results revealed that ERCC5 abnormal expression could influence the proliferation, apoptosis and migration ability of ovarian cancer cells by activating the Wnt pathway.