Apoptosis In Epithelial Ovarian Cancer Research Articles

THU542 Inhibiting Oxidosqualene Cyclase Suppresses Cancer Stem-like Cells And Growth Of Epithelial Ovarian Cancer Cells In Vitro And In Vivo

Abstract Disclosure: Y. Liang: None. K.P. Nephew: None. S.M. Hyder: None. Epithelial ovarian cancer (EOC) is an aggressive and deadly disease and despite concerted efforts to develop new strategies for preventing and treating the disease, almost 25,000 new cases are reported each year in the United States. Unfortunately, patients with ovarian cancer have poor prognosis due to the emergence of drug resistant cells and subsequent metastasis and have the highest mortality rate of all known gynecological malignancies. Standard treatments include systemic high-dose toxic chemotherapeutic drugs, but drug-resistance almost always occurs. Consequently, novel and more effective non-toxic therapies for ovarian cancer are urgently needed. The biosynthetic pathway leading to cholesterol production is an attractive therapeutic target since cholesterol is an essential structural and functional component of cell membranes and is necessary for tumor growth. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, alternative approaches to inhibit cholesterol biosynthesis are being considered. Epithelial ovarian cancer cells express enzymes in the cholesterol biosynthetic pathway. With this is mind we inhibited a key enzyme in the pathway, oxidosqualene cyclase (OSC), with a view to disrupting cholesterol biosynthesis and disrupting the progression of ovarian cancer. Potent small molecule inhibitors of OSC have been identified, and RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating breast and prostate cancer. Since OSC acts downstream of HMG-CoA reductase during cholesterol biosynthesis, its inhibition is not likely to be associated with the adverse effects caused by statins. We found in short-term assays (24-48 h) that pharmacological doses very effectively reduced the viability of ovarian tumor cells (SK-OV-3 and OVCAR-3), determined by Sulforhodamine B colorimetric assay. In long-term assays (7 days), nM concentrations of the drug were also effective. Importantly, RO (20-40 mg/kg/day; ip) significantly suppressed tumor xenografts derived from ovarian cancer cells (SK-OV-3; 336 + 60 mm3 vs 171 + 20 mm3) with no toxicity to experimental animals (27 treatments once tumors reached 100 mm3). Mechanistically, RO induced apoptosis in EOC, and suppressed ALDH activity, indicating that the inhibitor suppresses stem cell-like activity of EOC. Our study is the first to demonstrate disruption of cholesterol biosynthesis in human ovarian cancer cells as a novel and potent means by which to suppress their growth. Further studies will determine whether combining RO with chemotherapeutic drugs is an even more effective therapeutic approach to EOC, and examine the effects of RO on combating the development of drug-resistant ovarian cancer. Presentation: Thursday, June 15, 2023

Low expression of MYCN promotes cisplatin resistance by suppressing cisplatin-induced apoptosis in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most common cause of gynecological cancer-associated mortality. Cisplatin is one of the most effective chemotherapeutic drugs used in EOC; however, its use can lead to relapse due to cisplatin resistance. MYCN sensitizes neuroblastoma to undergo cisplatin-induced apoptosis. However, to the best of our knowledge, there have been no studies to date on the association between MYCN and cisplatin resistance in EOC. Therefore, the present study assessed this association. Datasets from The Cancer Genome Atlas database were used. The overall survival (OS) of patients receiving platin-based therapy was analyzed using Kaplan-Meier Plotter software. RNA sequencing data of 300 patients with EOC were downloaded from cBioportal. The co-expressed genes were subjected to 'Kyoto Encyclopedia of Genes and Genomes' analysis using DAVID software. For gene set enrichment analysis, the expression matrix was separated according to the median expression of MYCN, which was selected for hallmark gene set enrichment. Immunohistochemistry was used to assess MYCN expression in EOC tissue. Western blotting was used to evaluate MYCN, p53, Bax and Bcl-2 protein expression levels in EOC cells. Cell viability and apoptosis were assessed using Cell Counting Kit-8 and flow cytometry, respectively. The results demonstrated that MYCN upregulation was associated with increased cisplatin sensitivity and prolonged OS of patients with EOC and patients receiving platin-based therapy. Cisplatin downregulated MYCN expression in cisplatin-sensitive, but not resistant, EOC cells. The genes co-expressed with MYCN were primarily involved in pathways involved in 'chemotherapeutic resistance' and 'apoptosis'. MYCN enriched the apoptosis and p53 signaling pathways in hallmark gene sets. Cells in which MYCN was knocked down demonstrated significantly increased cisplatin resistance; however, MYCN overexpression in cisplatin-resistant cells restored cisplatin sensitivity. Collectively, the present study demonstrated that MYCN downregulation promoted cisplatin resistance by suppressing cisplatin-induced apoptosis in EOC.

The molecular mechanism of circRHOBTB3 inhibits the proliferation and invasion of epithelial ovarian cancer by serving as the ceRNA of miR-23a-3p

Rising evidences bespeak that circular RNAs are indispensable in regulating cellular biological behaviors and engaging in diseases' occurrence. CircRHOBTB3 has been reported to participate intimately in the progression of some cancers. Nevertheless, the mechanism by which circRHOBTB3 regulates tumorigenesis in epithelial ovarian cancer (EOC) remains ill-defined. The present study determined the expression pattern and bio-effects of circRHOBTB3 in EOC. Furthermore, it revealed that circRHOBTB3 could serve as the ceRNA of miR‑23a-3p to facilitate PTEN expression, suppress proliferation, G1/S transition, invasion, and promote apoptosis in EOC. Summarily, our findings provided a primary research foundation that circRHOBTB3 might be typified as a neoteric biomarker and a promising target of EOC, which is essential for improving the early diagnosis and precision treatment, so as to cut down EOC's mortality finally.

The transcription factor ZEB1 mediates the progression of epithelial ovarian cancer by promoting the transcription of CircANKRD17

Zinc finger E-box-binding homeobox 1 (ZEB1) is a key transcription factor that regulates the process of epithelial-mesenchymal transition (EMT) in various tumors. However, its role in epithelial ovarian cancer (EOC) is far from understood. The present study aimed to explore the role and potential mechanism of action of ZEB1 in EOC. A quantitativereverse transcription-polymerase chain reaction was used to detect ZEB1 expression levels. The Cell Counting Kit-8 assay, transwell assays, and flow cytometry were used to verify the effects of ZEB1 on the proliferation, invasion, migration, apoptosis, and EMT of EOC, respectively. RNAsequencing identified the effect of knocking down ZEB1 on circular RNAs in EOC cells. Dual-luciferase activity assay and chromatin immunoprecipitation experiments were used to verify the regulatory effect of ZEB1 on the circular RNA ANKRD17 (CircANKRD17; ID: hsa_circ_0007883) at the transcriptional level. Higher ZEB1 expression was found in EOC tissues and cells and was closely related to tumor metastasis, advanced stages, and lower survival rates. Furthermore, silencing ZEB1 inhibited the proliferation, invasion, migration, and EMT of EOC cells but enhanced cell apoptosis. Mechanistically, knockdown of ZEB1 resulted in the greatest downregulation of CircANKRD17 in EOC cells, and ZEB1 significantly promoted the expression of CircANKRD17 and had no significant effect on ANKRD17 messenger RNA expression. Further experiments verified that ZEB1 mediates the regulation of EOC processes by CircANKRD17. Highly expressed ZEB1 promoted proliferation, invasion, migration, and EMT while it inhibited cell apoptosis in EOC by promoting the transcription of CircANKRD17, providing a potential target for the treatment of EOC.

Calcineurin Gamma Catalytic Subunit PPP3CC Inhibition by miR-200c-3p Affects Apoptosis in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) outpaces all the other forms of the female reproductive system malignancies. MicroRNAs have emerged as promising predictive biomarkers to therapeutic treatments as their expression might characterize the tumor stage or grade. In EOC, miR-200c is considered a master regulator of oncogenes or tumor suppressors. To investigate novel miR-200c-3p target genes involved in EOC tumorigenesis, we evaluated the association between this miRNA and the mRNA expression of several potential target genes by RNA-seq data of both 46 EOC cell lines from Cancer Cell line Encyclopedia (CCLE) and 456 EOC patient bio-specimens from The Cancer Genome Atlas (TCGA). Both analyses showed a significant anticorrelation between miR-200c-3p and the protein phosphatase 3 catalytic subunit γ of calcineurin (PPP3CC) levels involved in the apoptosis pathway. Quantitative mRNA expression analysis in patient biopsies confirmed the inverse correlation between miR-200c-3p and PPP3CC levels. In vitro regulation of PPP3CC expression through miR-200c-3p and RNA interference technology led to a concomitant modulation of BCL2- and p-AKT-related pathways, suggesting the tumor suppressive role of PPP3CC in EOC. Our results suggest that inhibition of high expression of miR-200c-3p in EOC might lead to overexpression of the tumor suppressor PPP3CC and subsequent induction of apoptosis in EOC patients.

Periostin Secreted by Carcinoma-Associated Fibroblasts Promotes Ovarian Cancer Cell Platinum Resistance Through the PI3K/Akt Signaling Pathway.

Periostin (POSTN) is a protein secreted by mesenchymal cells. Periostin is upregulated in several cancer types and overexpression is associated with poor prognosis. However, the functional role and molecular underpinnings of periostin in epithelial ovarian cancer (EOC) is unknown. In the present study, periostin was found to be significantly upregulated in EOC stroma. Functional studies revealed that periostin could decrease cisplatin (DDP)-induced apoptosis in EOC. Periostin led to DDP resistance in EOC cells, potentially through the PI3K/Akt signaling pathway. We generated periostin-overexpressing fibroblasts and found that EOC cells were resistant to DDP when co-cultured with periostin-overexpressing fibroblasts. The findings of the present study indicated that periostin secreted by cancer-associated stromal cells may be a potential therapeutic target for EOC.

Circular RNA ABCB10 correlates with advanced clinicopathological features and unfavorable survival, and promotes cell proliferation while reduces cell apoptosis in epithelial ovarian cancer

This study aimed to explore the correlation of circular RNA ABCB10 (circ-ABCB10) expression with clinicopathological features and survival, as well as its impact on regulating cell proliferation and apoptosis in epithelial ovarian cancer (EOC). A total of 103 EOC patients were consecutively recruited, then their tumor tissues were obtained for circ-ABCB10 detection using qRT-PCR. Additionally, 53 EOC adjacent tissues were collected as control. Patients' clinicopathological and survival data were recorded. In vitro, circ-ABCB10 expression was detected in OVCAR3, UWB1.289, SKOV3, CAOV3 and IOSE80 cell lines by RT-qPCR, and the effect of circ-ABCB10 on cell proliferation and apoptosis was detected through circ-ABCB10 overexpression and silencing by plasmids transfection into SKOV3 cells. Circ-ABCB10 was upregulated in tumor tissues compared with adjacent tissues, and presented with good value in distinguishing tumor tissues from adjacent tissues (AUC = 0.766, 95% CI: 0.690-0.842). Circ-ABCB10 high expression was correlated with poor differentiation, large tumor size and advanced International Federation of Gynecology and Obstetrics (FIGO) stage in EOC patients. As for survival, circ-ABCB10 was correlated with worse OS. In vitro experiments revealed that circ-ABCB10 was upregulated and promoted cell proliferation but reduced cell apoptosis, and negatively regulated miR-1271, miR-1252 and miR-203 in EOC cells. Circ-ABCB10 correlates with advanced clinicopathological features and unfavorable survival, and promotes proliferation, reduces apoptosis and negatively regulated miR-1271, miR-1252 and miR-203 in EOC.

Induction of Apoptosis by Extract of Persian Gulf Marine Mollusk, Turbo Coronatus through the ROS-Mediated Mitochondrial Targeting on Human Epithelial Ovarian Cancer Cells.

Despite recent improvements in treatment, ovarian cancer is still the leading cause of death from gynaecological malignancies. Today, marine mollusks are considered as natural source of new biologically and pharmacologically active compounds by scientists and the pharmaceutical industries. The aim of this study is to investigate the selective apoptotic effects of Turbo coronatus crude extract fractions on human epithelial ovarian cancer (EOC) cells and mitochondria. The cells and mitochondria were isolated from cancerous and non-cancerous ovarian tissues and exposed to IC50 concentration of F1 fraction for evaluation of mitochondrial and cellular parameters. Our results showed that F1 fraction of T. coronatus crude extract significantly induced toxic effects only in the cancerous ovarian mitochondria, including increased reactive oxygen species (ROS) formation, mitochondrial membrane depolarization, mitochondrial swelling, and cytochrome c release.Flow-cytometry analysis demonstrated that F1 fraction of T. coronatus progressively induced apoptosis and necrosis only on EOC but not non-cancerous cells. We eventuallyconcluded that F1 fraction of T. coronatus crude extract selectively induces apoptosis in EOC through a ROS- mediated pathway.

Circ-ITCH correlates with small tumor size, decreased FIGO stage and prolonged overall survival, and it inhibits cells proliferation while promotes cells apoptosis in epithelial ovarian cancer.

The aim of this study was to evaluate the correlation of circular RNA itchy E3 ubiquitin protein ligase (Circ-ITCH) expression with clinicopathological features and survival in patients with epithelial ovarian cancer (EOC), and to explore its effect on cells proliferation as well as apoptosis in EOC cells. Seventy-seven EOC patients underwent surgery were retrospectively reviewed. Tumor tissues and paired adjacent tissues samples were collected, and Circ-ITCH expression was evaluated by quantitative polymerase chain reaction (qPCR). Blank mimic, Circ-ITCH mimic, blank inhibitor and Circ-ITCH inhibitor plasmids were transfected into SKOV3 cells and OVCAR-3 cells, and Counting Kit-8 (CCK-8) was performed to assess cells proliferation and Annexin V (AV)/propidium iodide (PI) was conducted to detect cells apoptosis. Median value of Circ-ITCH relative expression was 0.697 (0.367-1.106) in tumor tissues, which was lower compared with paired adjacent tissues (1.690 (0.867-2.813)) (P< 0.001), and it negatively correlated with tumor size (P= 0.005) as well as International Federation of Gynecology and Obstetrics (FIGO) stage (P< 0.001) in EOC patients. Multivariate Cox's analysis revealed that high Circ-ITCH expression was an independent predictive factor for favorable OS in EOC patients. Moreover, further in vitro experiments disclosed that Circ-ITCH expression was decreased in several EOC cell lines compared with normal ovarian epithelial cell line, and it inhibited cells proliferation while promoted cells apoptosis in SKOV3 and OVCAR-3 cells. Circ-ITCH correlates with small tumor size, decreased FIGO stage and prolonged OS, and it inhibits cells proliferation while promotes cells apoptosis in EOC.

Natural antisense transcript of hypoxia-inducible factor 1 regulates hypoxic cell apoptosis in epithelial ovarian cancer.

PurposeHypoxia is a key stress that triggers apoptosis in various tumors, including epithelial ovarian cancer (EOC). Previous researches identified a hypoxia-upregulated lncRNA named “a natural antisense transcript of hypoxia-inducible factor 1 (aHIF)” in some tumors. However, the contribution of aHIF to EOC remains unclear. Here, we aimed to investigate the expression, function, and underlying mechanisms of aHIF in EOC progression under hypoxia.Materials and methodsExpression levels of aHIF in EOC tissues were tested. In vitro and in vivo assays were conducted to explore the function and mechanism of aHIF in hypoxia-induced EOC progression.ResultsaHIF levels were increased in EOC tissues and were upregulated by hypoxia in EOC cells. Functional data revealed that aHIF knockdown accelerated cell apoptosis under hypoxia and inhibited EOC tumorigenesis and tumor growth in vivo. Additionally, aHIF overexpression inhibited cell apoptosis and enhanced cell proliferation under hypoxia in EOC. Mechanistically, the dysregulation of certain key mitochondrial apoptosis pathway-related genes, including Bcl-2, Bax, Caspase-7, and Caspase-9, may partially explain aHIF-regulated EOC apoptosis and growth under hypoxia.ConclusionThese data provide the first convincing evidence that aHIF may inhibit EOC apoptosis and thereby promote tumor growth through activation of the mitochondrial apoptosis pathway under hypoxia. Our findings help clarify the role of lncRNA in hypoxia-induced EOC progression.

Dihydroartemisinin induces apoptosis and inhibits proliferation, migration, and invasion in epithelial ovarian cancer via inhibition of the hedgehog signaling pathway.

Dihydroartemisinin (DHA), the primary of artemisinin extracted from the traditional Chinese medicine Artemisia annua, has been used in malaria treatment for a long time. Recently, many studies have indicated that, in addition to antimalarial effects, DHA also exhibits anticancer activity in certain types of neoplasms, including ovarian cancer. However, the precise anti‐ovarian cancer mechanism of DHA is still unclear. Abnormal activation of the hedgehog (Hh) pathway is closely related to tumorigenesis and progression of ovarian cancer. We performed this study to elucidate the effects of DHA on the biological behavior of ovarian cancer cells and to determine its effects on the Hh signaling pathway. CCK8 assays and flow cytometry were used to evaluate the effects of DHA on cell viability and apoptosis in both ovarian cancer cells and HOSEPICs (human ovarian surface epithelial cells) in response to DHA treatment. Transwell membrane chambers were used to analyze the effects of DHA on the migration and invasion of epithelial ovarian cancer cells following treatment with DHA. The impact of DHA on Hh signaling was analyzed by RT‐qPCR and Western blot. DHA significantly inhibited proliferation, migration, and invasion of ovarian cancer cells, and induced apoptosis in vitro. In contrast, DHA had few effects on cell proliferation and apoptosis in HOSEPICs. DHA inhibited the hedgehog signaling pathway. Furthermore, DHA inhibited purmorphamine (Hh signaling pathway agonist)‐induced cell proliferation, cell migration, and cell invasion and the inhibition of apoptosis. Importantly, DHA enhanced GANT61 (hedgehog signaling pathway inhibitor)‐induced apoptosis and the inhibition of cell viability, migratory capacity, and invasive ability. This study demonstrates that DHA inhibits cell viability, migration, and invasion, as well as induces apoptosis in epithelial ovarian cancer through suppression of the Hh signaling pathway.

Dicer affects cisplatin‑mediated apoptosis in epithelial ovarian cancer cells.

Dicer is an essential enzyme that processes micro (mi)-RNA precursors into mature miRNAs, and serves a critical role in cancer development and progression by regulating gene expression. However, the role of Dicer in cisplatin-mediated apoptosis and chemotherapy resistance in epithelial ovarian cancer (EOC) cells is poorly understood. In the present study, Dicer was expressed at low levels in cisplatin-resistant A2780 cells when compared with parental cells. In addition, knocking down Dicer using short hairpin RNA decreased the sensitivity of A2780 and CAOV3 cells to cisplatin. Furthermore, downregulating Dicer significantly inhibited cisplatin-induced apoptosis in ovarian cancer cells, and decreased the levels of proteins involved in apoptosis signaling pathways, including P73, P63, P53, caspase-9 and caspase-3. These findings indicated that Dicer may be a promising target for overcoming drug resistance in ovarian cancer.

Long non-coding RNA taurine-upregulated gene 1 predicts unfavorable prognosis, promotes cells proliferation, and inhibits cells apoptosis in epithelial ovarian cancer.

The aim of this study was to evaluate the correlation of long non-coding RNAs (lncRNAs) taurine-upregulated gene 1 (TUG1) with clinicopathological characteristics as well as overall survival (OS) in epithelial ovarian cancer (EOC) patients, and investigate its function in EOC cells proliferation and apoptosis in vitro.LncRNA TUG1 expressions were detected in tumor tissues and paired adjacent tissues obtained from 96 EOC patients. Blank mimic, lncRNA TUG1 mimic, blank inhibitor, and lncRNA TUG1 inhibitor plasmids were transfected into SKOV3 cells. CKK-8, annexin V-FITC–propidium iodide, qPCR and western blot assays were performed to detect cells proliferation, cells apoptosis, RNA expression, and protein expression, respectively.LncRNA TUG1 expression was higher in tumor tissue compared to paired adjacent tissue (P < .001), and it was positively correlated with pathological grade (P = .022), tumor size (P = .011) and FIGO stage (P < .001). Kaplan-Meier curve showed that lncRNA TUG1 high expression was associated with worse OS (P = .003). Multivariate Cox analysis indicated that lncRNA TUG1 high expression (vs. low expression) (P = .035) was independently predictive factor for shorter OS. In vitro, cells proliferation was promoted after treatment with lncRNA TUG1 mimic and was suppressed after treatment with lncRNA TUG1 inhibitor. In addition, cells apoptosis rate was decreased in lncRNA TUG1 mimic group compared to NC1 mimic, and increased in lncRNA TUG1 inhibitor group compared to NC2 inhibitor.In conclusion, lncRNA TUG1 is positively correlated with advanced disease and poor prognosis, and it promotes cells proliferation and inhibits cells apoptosis in EOC cells.

Adipose‑derived mesenchymal stem cells attenuate cisplatin‑induced apoptosis in epithelial ovarian cancer cells.

Environment-mediated drug resistance (EMDR) serves an important role in tumor chemotherapy resistance. Adipose‑derived mesenchymal stem cells (ADSC) are an important component of the tumor microenvironment. However, the role of ADSC in EMDR remains unclear. Therefore, in order to clarify whether ADSCs contribute to the cisplatin‑mediated apoptosis in epithelial ovarian cancer (EOC), the present study isolated ADSCs from the omentum of women with benign disease and collected the ADSC culture medium as conditioned medium. Subsequently, it was revealed that ADSCs decreased the sensitivity of EOC cells to cisplatin via an MTT assay. In addition, it was revealed that ADSCs may reduce cisplatin-induced apoptosis in EOC cells, as determined by Hoechst staining and flow cytometric analysis. Additionally, a lower level of cleaved caspase‑3 was observed, accompanied with decreased intracellular platinum accumulation in EOC cells indirectly co‑cultured with ADSCs. In conclusion, the findings of the present study suggested that ADSCs reduced cisplatin‑induced apoptosis and the intracellular level of platinum in EOC cells, which in turn resulted in cisplatin resistance. Therefore, ADSCs may serve as a therapeutic target for recurrent EOC.

Ubiquitin-specific protease 14 (USP14) regulates cellular proliferation and apoptosis in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Thus, there is an emergent need to invest a novel therapeutic for EOC. In this study, we defined ubiquitin-specific protease 14 (USP14) as a therapeutic target for EOC. Western blot was used to evaluate the expression of USP14 in nine fresh EOC tissues and three fresh normal ovarian tissues. The protein level of USP14 was higher in the cancer samples compared with that in the normal ovary tissues. Immunohistochemistry analysis was performed on formalin-fixed paraffin-embedded section of 116 cases of EOCs and indicated that USP14 was significantly associated with clinical pathologic variables. Kaplan-Meier curve showed that high expression of USP14 was related to poor prognosis of EOC patients. Starvation and re-feeding assay was used to imitate cell cycle, suggesting that USP14 played a critical role in SKOV3 cell proliferation. CCK-8 assay showed that SKOV3 cells treated with USP14-shRNA (shUSP14) grew more slowly than control group. Flow cytometry revealed that the reduced expression of USP14 induced the apoptosis of the SKOV3 EOC cells. In summary, our findings suggest that USP14 is involved in the progression of EOC and that it may be a useful target of therapy in EOC.

Abstract 526: Combined inhibition of mTOR and AKT activity induces potent apoptosis in epithelial ovarian cancer cells .

Abstract Mammalian target of rapamycin (mTOR) and Phosphatidylinositol 3-kinase (PI3K) are two key components of the PI3K/Akt/mTOR signaling pathway. These two signal transduction cascades regulate a number of physiological cell functions including differentiation, proliferation, apoptosis and autophagy. Dysregulation of these pathways have been found in many cancers including DLBCL, thyroid cancer, breast cancer, prostate cancer and lung cancer. mTOR has been shown to activate the PI3-kinase/AKT survival pathway and understanding the interplay among signaling molecules in the PI3K/Akt/mTOR pathway is of utmost importance. Two distinct mTOR complexes; mTORC1 and mTORC2, have been identified. mTORC1 is downstream of Akt, sensitive to rapamycin inhibition, and controls cap-dependent protein translation. In contrast, mTORC2 is directly upstream of Akt and is resistant to rapamycin. In this study, we investigated the effect of mTOR inhibition on cell viability and induction of apoptosis in epithelial ovarian cancer (EOC) cells, either alone or in combination with PI3-kinase/AKT inhibitor. We found that Torin2, an mTOR inhibitor, decreased cell viability and induced apoptosis in EOC cell lines in a dose dependent manner. Torin2 also inactivated mTOR1 and mTOR2 as well as downstream targets of mTOR; 4E-BP1 and p70S6. Torin2 treatment of EOC cell lines also induced mitochondrial dependent apoptosis via activation and cleavage of caspases. Finally, combination treatment of EOC cells with Torin2 and LY294002, a PI3-kinase/AKT inhibitor induced a synergistic apoptotic response via inactivation of AKT, mTOR1 and mTOR2 and activation of caspases. These studies will give us an insight on the molecular relationship between these survival pathways and suggest that simultaneous inhibition of mTOR and PI3-kinase/AKT pathways may be beneficial for the management of EOC. Citation Format: Azhar R. Hussain, Shahab Uddin, Maqbool Ahmed, Khawla S. Al-Kuraya. Combined inhibition of mTOR and AKT activity induces potent apoptosis in epithelial ovarian cancer cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 526. doi:10.1158/1538-7445.AM2013-526

Role of 12-lipoxygenase in regulation of ovarian cancer cell proliferation and survival

Eicosanoid-related enzymes have been implicated in the pathogenesis of various cancers. Little is known about the relevance of lipoxygenase pathway to ovarian cancer growth. In this study, we examined the role of 12-lipoxygenase (12-LOX), the main human 12-HETE generating enzyme, in the regulation of proliferation and survival in epithelial ovarian cancer. Immunohistological analysis of 12-LOX expression in high-grade serous ovarian carcinoma and normal ovarian epithelium tissues was performed. The presence of 12-LOX-12-HETE system was confirmed in two epithelial ovarian cancer (EOC) cell lines, OVCAR-3 and SK-OV-3, using RT-PCR, Western blot and LC/MS analysis. The effects of N-benzyl-N-hydroxy-5-phenyl-pentanamide (BMD-122), a specific 12-LOX inhibitor, on cell growth, survival, apoptosis, and cell signaling were determined. We found that a significantly higher level of 12-LOX expression in high-grade serous ovarian carcinoma compared to normal ovarian epithelium. OVCAR-3 and SK-OV-3 were found to express high level of 12-LOX mRNA and protein. Both EOC increased their 12-HETE production when incubated with arachidonic acid. BMD-122 inhibited the EOC growth in a dose-dependent fashion. Purified 12-HETE significantly reversed such inhibitory effects of BMD-122. In addition, BMD-122 blocked the MAPK signaling pathway by inhibiting the phosphorylation of ERK and induced a ~20-30% increase in the EOC apoptosis. Down-regulation of the 12-LOX expression using 12-LOX siRNA also resulted in markedly reduction in cell growth. These data suggest that 12-LOX is involved in the regulation of ovarian cancer cell growth and survival and is a potential new therapeutic target.

Myeloperoxidase serves as a redox switch that regulates apoptosis in epithelial ovarian cancer

Objectives Resistance to apoptosis is a key feature of cancer cells and is believed to be regulated by nitrosonium ion (NO +)-induced S-nitrosylation of key enzymes. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is utilized by MPO to generated NO +. We sought to investigate the expression of myeloperoxidase (MPO) and iNOS in epithelial ovarian cancer (EOC) and determine their effect on S-nitrosylation of caspase-3 and its activity as well as apoptosis. Methods MPO and iNOS expression were determined using immunofluorescence in SKOV-3 and MDAH-2774 and EOC tissue sections. S-nitrosylation of caspase-3 and its activity, levels of MPO and iNOS, as well as apoptosis, were evaluated in the EOC cells before and after silencing MPO or iNOS genes with specific siRNA probes utilizing real-time RT–PCR, ELISA, and TUNEL assays. Results MPO and iNOS are expressed in EOC cell lines and in over 60% of invasive EOC cases with no expression in normal ovarian epithelium. Indeed, silencing of MPO or iNOS gene expression resulted in decreased S-nitrosylation of caspase-3, increased caspase-3 activity, and increased apoptosis but with a more significant effect when silencing MPO. Conclusion MPO and iNOS are colocalized to the same cells in EOC but not in the normal ovarian epithelium. Silencing of either MPO or iNOS significantly induced apoptosis, highlighting their role as a redox switch that regulates apoptosis in EOC. Understanding the mechanisms by which MPO functions as a redox switch in regulating apoptosis in EOC may lead to future diagnostic tools and therapeutic interventions.

The effects of combining docetaxel and cyclooxygenase-2 inhibitors on proliferation and apoptosis in epithelial ovarian cancer

In-vitro studies have shown that taxanes can upregulate cellular cyclooxygenase-2 expression. The purpose of this study is to evaluate the effects of the combination, cyclooxygenase-2 inhibitor and docetaxel, on epithelial ovarian cancer cells. Four epithelial ovarian cancer cell lines (MDAH-2774, SKOV3, OVCAR and CaOV-3) were treated with the specific cyclooxygenase-2 inhibitor NS398 (10 or 100 mumol/l) and docetaxel (0.1, 1 or 10 mumol/l) in various combinations. Apoptosis in the ovarian cancer cells was assessed using TUNEL assay. Multiplex reverse transcription-PCR was used to determine mRNA levels of cyclooxygenase-2, bcl-2 and bax. Treatment of all epithelial ovarian cancer cells with docetaxel resulted in significant apoptotic death. Concurrent treatment of MDAH-2774, SKOV3 and OVCAR cells with docetaxel and NS398 resulted in the reduction of the taxane-induced apoptosis. Similar reduction was seen when the cells were exposed to NS398 for 4 h before docetaxel treatment. Conversely, treating the MDAH-2774 and SKOV3 cells with docetaxel followed by NS398 resulted in a significant increase in apoptosis compared with treatment with the taxane alone. bax mRNA levels were significantly reduced in SKOV3 cells treated concurrently with NS398 and docetaxel; bcl-2 mRNA levels showed no change. When combining docetaxel and a cyclooxygenase-2 inhibitor in the treatment of ovarian cancer cells, the sequencing of the drugs seems to have an important influence on the overall outcome. Using the cyclooxygenase-2 inhibitor before or concurrently with the taxane will result in a reduction of cellular apoptotic death. This might be due to a reduction in the expression of the proapototic gene bax.

Neoadjuvant chemotherapy for epithelial ovarian cancer—role of apoptosis

Ovarian cancer is one of the most frequently fatal gynecological cancers because most cases are diagnosed at an advanced stage. Loss of growth control and a marked resistance to apoptosis are considered major mechanisms driving tumor progression. Little is known about the effect of various treatment regimens on the distribution of molecular markers of apoptosis in epithelial ovarian cancer. The objective of this study was to compare the expression levels of both proapoptotic and antiapoptotic proteins p53, p73, Bcl-2, Bcl-XL and survivin in the ascitic cells and tumor samples of patients undergoing treatment with two different regimens. A total of 24 patients with untreated epithelial ovarian cancer were randomized into two groups of 12 each. Group 1 patients received three cycles of chemotherapy prior to surgery and three cycles after surgery and group 2 patients received six cycles of chemotherapy prior to surgery. The expression of apoptosis-related proteins was analyzed in ascitic fluid and tumor samples by Western blotting and immunohistochemistry. The apoptotic index was also determined in these samples by the TUNEL assay. Significant decreases in antiapoptotic bcl-2 and survivin were seen, accompanied by increases in apoptotic index in tumors that had undergone chemotherapy as compared to the baseline ascites samples. No significant change in bcl-XL was observed. A significant decrease in proapoptotic p53 was also seen. No expression of p73 was observed in tumors or ascites. The findings were similar in groups 1 and 2 patients and were not statistically significantly different, perhaps due to the small sample size (n=12) of each group. The above findings indicate that chemotherapy in ovarian carcinoma leads to an increase in apoptosis by a p53-independent pathway, which involves the downregulation of antiapoptotic Bcl-2 and survivin but not Bcl-XL. Furthermore, administering neoadjuvant chemotherapy (six cycles) as an alternative form of therapy for advanced epithelial ovarian cancer is more effective in inducing apoptosis than three cycles. However, the findings of this study need to be corroborated using a larger sample.