Clear Cell Carcinoma Cell Research Articles

Preclinical Investigations of PM01183 (Lurbinectedin) as a Single Agent or in Combination with Other Anticancer Agents for Clear Cell Carcinoma of the Ovary.

ObjectiveThe objective of this study was to evaluate the antitumor effects of lurbinectedin as a single agent or in combination with existing anticancer agents for clear cell carcinoma (CCC) of the ovary, which is regarded as an aggressive, chemoresistant, histological subtype.MethodsUsing human ovarian CCC cell lines, the antitumor effects of lurbinectedin, SN-38, doxorubicin, cisplatin, and paclitaxel as single agents were assessed using the MTS assay. Then, the antitumor effects of combination therapies involving lurbinectedin and 1 of the other 4 agents were evaluated using isobologram analysis to examine whether these combinations displayed synergistic effects. The antitumor activity of each treatment was also examined using cisplatin-resistant and paclitaxel-resistant CCC sublines. Finally, we determined the effects of mTORC1 inhibition on the antitumor activity of lurbinectedin-based chemotherapy.ResultsLurbinectedin exhibited significant antitumor activity toward chemosensitive and chemoresistant CCC cells in vitro. An examination of mouse CCC cell xenografts revealed that lurbinectedin significantly inhibits tumor growth. Among the tested combinations, lurbinectedin plus SN-38 resulted in a significant synergistic effect. This combination also had strong synergistic effects on both the cisplatin-resistant and paclitaxel-resistant CCC cell lines. Everolimus significantly enhanced the antitumor activity of lurbinectedin-based chemotherapies.ConclusionsLurbinectedin, a new agent that targets active transcription, exhibits antitumor activity in CCC when used as a single agent and has synergistic antitumor effects when combined with irinotecan. Our results indicate that lurbinectedin is a promising agent for treating ovarian CCC, both as a first-line treatment and as a salvage treatment for recurrent lesions that develop after platinum-based or paclitaxel treatment.

High-level secretion of tissue factor-rich extracellular vesicles from ovarian cancer cells mediated by filamin-A and protease-activated receptors.

Thromboembolic events occur frequently in ovarian cancer patients. Tissue factor (TF) is often overexpressed in tumours, including ovarian clear-cell carcinoma (CCC), a subtype with a generally poor prognosis. TF-coagulation factor VII (fVII) complexes on the cell surface activate downstream coagulation mechanisms. Moreover, cancer cells secrete extracellular vesicles (EVs), which act as vehicles for TF. We therefore examined the characteristics of EVs produced by ovarian cancer cells of various histological subtypes. CCC cells secreted high levels of TF within EVs, while the high-TF expressing breast cancer cell line MDA-MB-231 shed fewer TF-positive EVs. We also found that CCC tumours with hypoxic tissue areas synthesised TF and fVII in vivo, rendering the blood of xenograft mice bearing these tumours hypercoagulable compared with mice bearing MDA-MB-231 tumours. Incorporation of TF into EVs and secretion of EVs from CCC cells exposed to hypoxia were both dependent on the actin-binding protein, filamin-A (filA). Furthermore, production of these EVs was dependent on different protease-activated receptors (PARs) on the cell surface. These results show that CCC cells could produce large numbers of TF-positive EVs dependent upon filA and PARs. This phenomenon may be the mechanism underlying the increased incidence of venous thromboembolism in ovarian cancer patients.

Lipocalin 2 attenuates iron-related oxidative stress and prolongs the survival of ovarian clear cell carcinoma cells by up-regulating the CD44 variant

ABSTRACTOvarian clear cell carcinoma (CCC) arises from ovarian endometriosis. Intra-cystic fluid contains abundant amounts of free iron, which causes persistent oxidative stress, a factor that has been suggested to induce malignant transformation. However, the mechanisms linking oxidative stress and carcinogenesis in CCC currently remain unclear. Lipocalin 2 (LCN2), a multifunctional secretory protein, functions as an iron transporter as well as an antioxidant. Therefore, we herein examined the roles of LCN2 in the regulation of intracellular iron concentrations, oxidative stress, DNA damage, and antioxidative functions using LCN2-overexpressing (ES2), and LCN2-silenced (RMG-1) CCC cell lines. The results of calcein staining indicated that the up-regulated expression of LCN2 correlated with increases in intracellular iron concentrations. However, a DCFH-DA assay and 8OHdG staining revealed that LCN2 reduced intracellular levels of reactive oxygen species and DNA damage. Furthermore, the expression of LCN2 suppressed hydrogen peroxide-induced apoptosis and prolonged cell survival, suggesting an antioxidative role for LCN2. The expression of mRNAs and proteins for various oxidative stress-catalyzing enzymes, such as heme oxygenase (HO), superoxide dismutase (SOD), and glutathione peroxidase, was not affected by LCN2, whereas the intracellular concentration of the potent antioxidant, glutathione (GSH), was increased by LCN2. Furthermore, the expression of xCT, a cystine transporter protein, and CD44 variant 8-10 (CD44v), a stem cell marker, was up-regulated by LCN2. Although LCN2 increased intracellular iron concentrations, LCN2-induced GSH may catalyze and override oxidative stress via CD44v and xCT, and subsequently enhance the survival of CCC cells in oxidative stress-rich endometriosis.

Delphinidin suppresses proliferation and migration of human ovarian clear cell carcinoma cells through blocking AKT and ERK1/2 MAPK signaling pathways

Delphinidin possesses the highest chemopreventive activity among the six components of anthocyanidin that are pigments from fruits and vegetables giving them blue, purple or red colors. Although delphinidin has anti-carcinogenic and apoptotic effects in various cancers, little is known about its functional roles in ovarian clear cell carcinoma (CCC) which shows poor prognosis with resistance to chemotherapy as compared with other subtypes of epithelial ovarian cancers (EOC). Results of present study revealed that cell survival rates of ES2 cells from ovarian CCC treated with delphinidin decreased in a dose-dependent manner. Also, delphinidin inhibited migration and induced apoptosis of ES2 cells. To investigate the molecular mechanisms responsible for biological effects of delphinidin, we analyzed the phosphorylation status of carcinogenic protein kinases related to development of CCC in a dose- and time-dependent manner. Phosphorylation of downstream targets of PI3K (AKT and p70S6K) and MAPKs (ERK1/2 and JNK) signaling was suppressed by treatment of ES2 cells with delphinidin. In addition, pharmacological inhibitors of PI3K/AKT and ERK1/2 MAPK pathway improved the anti-proliferative action of delphinidin on ES2 cells. Moreover, we compared the cancer preventive effects of delphinidin with traditional cisplatin- and paclitaxel-based chemotherapy on cell viability of ES2 cells. Results showed that delphinidin is as effective in its therapeutic activity against ES2 cells as cisplatin and placlitaxel. Collectively, these results indicated that delphinidin plays a critical role as a new chemotherapeutic agent to prevent development and progression of ES2 cells in CCC via inactivation of PI3K/AKT and ERK1/2 MAPK signal transduction cascades.

Identification of intranuclear inclusions is useful for the cytological diagnosis of ovarian clear cell carcinoma.

The aim of this study was to clarify the diagnostic significance of the presence of intranuclear inclusions in clear cell carcinoma (CCC). We analyzed 98 imprint specimens and 53 ascites specimens from 98 ovarian carcinoma cases [28 CCCs, 37 serous carcinomas (SCs), 22 endometrioid carcinomas (ECs), and 11 mucinous carcinomas (MCs)]. We examined (1) frequency of intranuclear inclusion-positive cases of each ovarian carcinoma subtype, using imprint specimens, (2) frequency of intranuclear inclusion-positive cells of each ovarian carcinoma subtype, using imprint specimens, (3) frequency of intranuclear inclusion-positive cases of each ovarian carcinoma subtype, using ascites specimens, and (4) sensitivity and specificity of the presence of intranuclear inclusions for the cytological diagnosis of CCC. (1) The frequency of intranuclear inclusion-positive cases in CCC (96.4%) was significantly higher than in SC (13.5%), EC (13.6%), and MC (18.2%) (P < 0.001). Two or more intranuclear inclusions in a single nucleus were observed only in CCC. (2) The frequency of intranuclear inclusion-positive cells in CCC (median, 0.41%) was significantly higher than in non-CCC subtypes (0.010%) (P < 0.001). (3) Using ascites specimens, the frequency of intranuclear inclusion-positive cases in CCC (78.6%) was significantly higher than in SC (10.3%), EC (0%), and MC (0%) (P < 0.001). (4) The sensitivity of intranuclear inclusions was 96.4%, and the specificity was 85.7%. The identification of intranuclear inclusions, in particular a high frequency and multiple intranuclear inclusions in a single nucleus, is useful for the cytological diagnosis of CCC. Furthermore, these results may be applicable to ascites cytology.

Anti-apoptotic genes are synergistically activated in OVSAYO cells cultured under conditions of serum starvation and hypoxia.

The tumor microenvironment is generally hypoxic because of the limited oxygen supply from inefficient or insufficient vasculature. Hypoxic tumor tissues are also poorly supplied with serum components. We have previously demonstrated that expression of the FVII gene is induced in response to hypoxia in ovarian clear cell carcinoma (CCC) cells. This gene activation is synergistically enhanced when cells are simultaneously subjected to serum starvation, and is dependent on the transcription factor Sp1 directly associating with the FVII promoter. We have identified additional genes activated via a similar Sp1-dependent mechanism by conducting cDNA microarray analysis (GSE55565). ICAM1, which encodes intercellular adhesion molecule-1 (ICAM-1), is one such gene. ICAM-1 confers an anti-apoptotic effect upon CCC cells in vitro and promotes growth of CCC tumors. Here we describe the transcriptome analysis performed in our recently published study (Koizume et al., 2015). We further show that autonomous activation of the TNFα–NFκB axis is responsible for the synergistic activation of ICAM1 under hypoxic and serum starvation conditions. This study provides additional information as to how CCC cell survival can be facilitated under conditions of serum starvation and hypoxia.

Fibroblast growth factor receptor 2 is associated with poor overall survival in clear cell carcinoma of the ovary and may be a novel therapeutic approach.

We previously found that gene and protein expression of fibroblast growth factor receptor (FGFR) 2 were increased in ovarian clear cell carcinoma (CCC); here, we examined FGFR2 expression in CCC tumor tissues and its correlation with clinical parameters. We also analyzed the effect of an FGFR inhibitor on the growth of CCC cells to investigate whether FGFR2 could be a therapeutic target for this disease. We analyze the protein expression of FGFR2 by immunohistochemical staining in CCC from 112 patients and evaluated the association of these molecular parameters with clinical outcome. We treated the 11 CCC cell lines with an FGFR inhibitor, and then assessed cell viability, the expression of protein in FGFR2 signaling pathway, and cell cycle distribution. The expressions of FGFR2 were found in 96% of CCC. The 5-year survival rate for patients with a moderate or strong expression of FGFR2 was significantly lower than that for those with an absent or poor expression of FGFR2 (54% vs 79%). Multivariable analysis revealed that FGFR2 expression and disease stage were independent prognostic factors. The FGFR inhibitor effectively suppressed the growth of CCC cells with induction of G1 cell cycle arrest and down-regulated the expression of phosphorylated Akt and phosphorylated ERK. FGFR2 is an important biomarker predictive of patient outcome and is a potential target for CCC. Further study is warranted for FGFR inhibitor to treat CCC.

Lipid starvation and hypoxia synergistically activate ICAM1 and multiple genes in an Sp1-dependent manner to promote the growth of ovarian cancer.

BackgroundElucidation of the molecular mechanisms by which cancer cells overcome hypoxia is potentially important for targeted therapy. Complexation of hypoxia-inducible factors (HIFs) with aryl hydrocarbon receptor nuclear translocators can enhance gene expression and initiate cellular responses to hypoxia. However, multiple molecular mechanisms may be required for cancer cells to adapt to diverse microenvironments. We previously demonstrated that a physical interaction between the ubiquitously expressed transcription factor Sp1 and HIF2 is a major cause of FVII gene activation in poor prognostic ovarian clear cell carcinoma (CCC) cells under hypoxia. Furthermore, it was found that FVII activation is synergistically enhanced when serum-starved cells are cultured under hypoxic conditions. In this study, we investigated whether HIFs and transcription factor Sp1 cooperate to activate multiple genes in CCC cells under conditions of serum starvation and hypoxia (SSH) and then contribute to malignant phenotypes.MethodsTo identify genes activated under hypoxic conditions in an Sp1-dependent manner, we first performed cDNA microarray analyses. We further investigated the molecular mechanisms of synergistic gene activations including the associated serum factors by various experiments such as real-time RT-PCR, western blotting and chromatin immunoprecipitation. The study was further extended to animal experiments to investigate how it contributes to CCC progression in vivo.ResultsICAM1 is one such gene dramatically induced by SSH and is highly induced by SSH and its synergistic activation involves both the mTOR and autonomously activated TNFα-NFκB axes. We identified long chain fatty acids (LCFA) as a major class of lipids that is associated with albumin, a serum factor responsible for synergistic gene activation under SSH. Furthermore, we found that ICAM1 can be induced in vivo to promote tumor growth.ConclusionSp1 and HIFs collaborate to activate genes required for the adaptation of CCC cells to severe microenvironments, such as LCFA starvation and hypoxia. This study highlights the importance of transcriptional regulation under lipid starvation and hypoxia in the promotion of CCC tumor growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0351-z) contains supplementary material, which is available to authorized users.

Preclinical Efficacy for AKT Targeting in Clear Cell Carcinoma of the Ovary

The aim of this study was to determine the role of AKT as a therapeutic target in ovarian clear cell carcinoma (CCC), an aggressive, chemoresistant histologic subtype of ovarian cancer. AKT activation was assessed by immunohistochemistry (IHC) using human tissue microarrays of primary ovarian cancers, composed of both CCC and serous adenocarcinoma (SAC). The growth-inhibitory effect of AKT-specific targeting by the small-molecule inhibitor, perifosine, was examined using ovarian CCC cell lines in vitro and in vivo. Finally, the activity of perifosine was examined using in CCC-derived tumors that had acquired resistance to anti-VEGF or chemotherapeutics such as bevacizumab or cisplatin, respectively. Interestingly, AKT was frequently activated both in early-stage and advanced-stage CCCs. Treatment of CCC cells with perifosine attenuated the activity of AKT-mTORC1 signaling, inhibited proliferation, and induced apoptosis. The effect of perifosine was more profound under conditions of high AKT activity compared with low AKT activity. Increased AKT activation and enhanced sensitivity to perifosine were observed in the context of cisplatin-resistant CCC. Treatment with perifosine concurrently with cisplatin significantly enhanced the antitumor effect of cisplatin. Moreover, perifosine showed significant antitumor activity in CCC-derived tumors that had acquired resistance to bevacizumab or cisplatin. Collectively, these data reveal that AKT is frequently activated in ovarian CCCs and is a promising therapeutic target in aggressive forms of ovarian cancer. AKT-targeted therapy has value in a first-line setting as well as a second-line treatment for recurrent disease developing after platinum-based chemotherapy or bevacizumab treatment.

Cystatin B as a potential diagnostic biomarker in ovarian clear cell carcinoma.

Epithelial ovarian cancer (EOC) consists of four major subtypes: clear cell carcinoma (CCC), endometrioid adenocarcinoma (EA), mucinous adenocarcinoma (MA) and serous adenocarcinoma (SA). Relative to the other subtypes, the prognosis of CCC is poor due to a high recurrence rate and chemotherapy resistance, but CCC-specific biomarkers have yet to be identified. With the aim of identifying diagnostic and treatment biomarkers for CCC, we analyzed 96 cases of EOC (32 CCC, 13 EA, 19 MA, 32 SA) using liquid chromatography/mass spectrometry (LC/MS) followed by immunohistochemistry (IHC) and quantitative reverse transcription PCR (RT-qPCR). Semi-quantification of protein differences between subtypes showed upregulation of 150 proteins and downregulation of 30 proteins in CCC relative to the other subtypes. Based on hierarchical clustering that revealed a marked distinction in the expression levels of cystatin B (CYTB) and Annexin A4 (ANXA4) in CCC relative to the other subtypes, we focused the study on CYTB and ANXA4 expression in EOCs by IHC, RT-qPCR and western blot analyses using tissue specimens and cultured cells. As a result, compared to the other subtypes, CCC showed significantly high expression levels of CYTB and ANXA4 in the analyses. To examine the possibility of CYTB and ANXA4 as serum diagnostic biomarkers of CCC, we checked the protein levels in conditioned media and cell lysates using culture cells. Compared with the other subtypes, CCC cell lines showed a significantly higher level of expression of CYTB in both conditioned media and cell lysates, while ANXA4 showed a higher level of expression in cell lysates only. Our results demonstrate that CYTB and ANXA4 overexpression may be related to carcinogenesis and histopathological differentiation of CCC. CYTB may be a secreted protein, and may serve as a potential serum diagnostic biomarker of CCC, while ANXA4 may be useful as an intracellular marker.

MicroRNA-21 is a candidate driver gene for 17q23-25 amplification in ovarian clear cell carcinoma.

BackgroundEpithelial ovarian cancer (EOC) is the most common cause of gynecological malignancy-related mortality. Ovarian clear cell carcinoma (CCC) has unique clinical characteristics and behaviors that differ from other histological types of EOC, including a frequent association with endometriosis and a highly chemoresistant nature, resulting in poor prognosis. However, factors underlying its malignant behavior are still poorly understood. Aberrant expression of microRNAs has been shown to be involved in oncogenesis, and microRNA-21 (miR-21) is frequently overexpressed in many types of cancers. The aim of this study was to investigate the role of miR-21 in 17q23-25 amplification associated with CCC oncogenesis.MethodsWe identified 17q23-25 copy number aberrations among 28 primary CCC tumors by using a comparative genomic hybridization method. Next, we measured expression levels of the candidate target genes, miR-21 and PPM1D, for 17q23-25 amplification by real-time RT-PCR analysis and compared those data with copy number status and clinicopathological features. In addition, immunohistochemical analysis of PTEN (a potential target of miR-21) was performed using the same primary CCC cases. We investigated the biological significance of miR-21 overexpression in CCC using a loss-of-function antisense approach.Results17q23-25 amplification with both miR-21 overexpression and PTEN protein loss was detected in 4/28 CCC cases (14.2%). The patients with 17q23-25 amplification had significantly shorter progression-free and overall survival than those without 17q23-25 amplification (log-rank test: p = 0.0496; p = 0.0469, respectively). A significant correlation was observed between miR-21 overexpression and endometriosis. Both PTEN mRNA and PTEN protein expression were increased by miR-21 knockdown in CCC cells. We also confirmed that miR-21 directly bound to the 3′-untranslated region of PTEN mRNA using a dual-luciferase reporter assay.ConclusionsMiR-21 is a possible driver gene other than PPM1D for 17q23-25 amplification in CCC. Aberrant expression of miR-21 by chromosomal amplification might play an important role in CCC carcinogenesis through the regulation of the PTEN tumor suppressor gene.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-799) contains supplementary material, which is available to authorized users.

Abstract 502: Sp1 and HIFs mediate synergistic activation of ICAM1 gene under serum starved hypoxia and promotes growth of ovarian cancer

Abstract Cancer tissues are generally exposed to severe hypoxia conditions due to sparse and aberrant vasculature. Hypoxia-inducible factors HIF-1α and HIF-2α (HIF1 and HIF2, respectively) are major transcription factors required for adaptive responses to hypoxia. HIFs complexed with arylhydrocarbon receptor nuclear translocator bind target genes and act as enhancer of transcriptional regulation. This conventional mechanism regulates genes such as VEGF, GLUT1, and HO to improve mild to severe hypoxia conditions. It is likely that various oxygen concentrations and their duration exist within tumor tissue. It appears that adaptive response by HIFs is a major way to overcome stresses under milder hypoxia. However, majority of tumor tissues of many cancer types are known to undergo more severe hypoxic conditions. Thus, additional molecular mechanisms supporting HIFs signaling may be required to enhance survival activity of cancer cells. We have previously demonstrated that FVII gene, which is essential for initiation of extrinsic blood coagulation can be induced in ovarian clear cell carcinoma (CCC) cells in response to hypoxia. Activation of this gene potentially results in thrombotic events frequently observed in cancer patients. We have demonstrated that a physical interaction between Sp1 and HIF2 is a major cause of the activation of this gene under hypoxia. Furthermore, FVII activation is synergistically enhanced when cells are cultured under ischemic condition, in which hypoxia and serum-free conditions coexist. We envisioned that genes regulated by this mechanism are responsible for adaptive response to hypoxia with severity and/or duration characteristic to CCC tissues. Furthermore, ovarian cancer is a most malignant among gynecological diseases. CCC is highly tolerant to chemotherapy and the prognosis is poor. Thus, we first explored genes those are regulated by both Sp1 and HIFs in CCC cells by cDNA microarray analysis. We found that Sp1 and hypoxia inducible factors activate multiple genes under the ischemic condition in CCC cells. Among genes which meet this criteria, ICAM1 is one of a most dramatically induced gene. This transcriptional induction involves activation of NFκB associated with mTOR and autonomously produced TNFα. We further performed xenograft tumor experiments to test whether ICAM1 can be induced and contributes to CCC progression in vivo. It was found that ICAM1 is dramatically induced within xenograft tumor tissues. Tumor growth was evidently suppressed when ICAM1 inducton was abrogated by shRNA transfection. Thus, interplay among transcription factors associated with serum deficiency results in cancer progression. This study highlights importance of adaptive response to serum-starved condition as to how cancer cells overcome severe hypoxic stress. Citation Format: Shiro Koizume, Shin Ito, Yoshiyasu Nakamura, Mitsuyo Yoshihara, Yasuo Takano, Yohei Miyagi. Sp1 and HIFs mediate synergistic activation of ICAM1 gene under serum starved hypoxia and promotes growth of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 502. doi:10.1158/1538-7445.AM2014-502

Abstract 3024: Cancer stem-like cells in ovarian clear cell carcinoma are enriched in ALDH-high population associated with the accelerated scavenging system of reactive oxygen species

Abstract In ovarian cancer cases, disease recurrence after chemotherapy is frequently observed, suggesting ovarian cancer stem-like cells (CSCs) are involved. Ovarian clear cell carcinoma is especially chemo-resistant among epithelial ovarian cancer subtypes. We first, examined the CSC markers (CD133,CD44, ALDH1) using the ovarian clear cell carcinoma tissues (n=81). Immunohistochemical staining revealed that high ALDH1 expression levels are related to advanced stage in clear cell carcinoma cases. Other makers are not related to clinical staging and prognosis. Clear cellcarcinoma cells (KOC-7C) are separated to ALDH-high and ALDH-low population by ALDEFLUOR assay and fluorescence-activated cell sorting. ALDH-high cells showed more quiescent character (G0/G1 phase) than ALDH-low cells. Recent studies have suggested that CSCs might have a high antioxidant capacity to keep cellular reactive oxygen species (ROS) at a low level that allows them to survive adverse conditions. We examined the cellular ROS level using Cell-ROX assay. ALDH-high cells contained the lower levels of ROS than ALDH-low cells. Also, ALDH-high cells showed an increased expression of in nuclear factor-E2-related factor 2 (Nrf2), a key transcriptional factor of the antioxidant system, associated with the increased antioxidant enzymes (SOD2, HO-1). Thus, ALDH-positive CSCs might have increased Nrf2-induced antioxidant scavengers, which lower ROS level relevant to chemo-resistance in ovarian clear cell carcinoma. Citation Format: Tomoko Mizuno, Noriko Suzuki, Minako Mori, Hiroshi Makino, Tatsuro Furui, Naoki Ito, Akio Yamamoto, Kennichiro Morishige. Cancer stem-like cells in ovarian clear cell carcinoma are enriched in ALDH-high population associated with the accelerated scavenging system of reactive oxygen species. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3024. doi:10.1158/1538-7445.AM2014-3024

Selective cytotoxicity of indirect nonequilibrium atmospheric pressure plasma against ovarian clear-cell carcinoma.

Ovarian clear cell carcinoma (CCC) is a histological type of epithelial ovarian cancer that is less responsive to chemotherapy and associated with a poorer prognosis than serous and endometrioid carcinoma. Non-thermal atmospheric pressure plasma which produces reactive species has recently led to an explosion of research in plasma medicine. Plasma treatment can be applied to cancer treatment to induce apoptosis and tumor growth arrest. Furthermore, recent studies have shown that a medium exposed to plasma also has an anti-proliferative effect against cancer in the absence of direct exposure to plasma. In this study, we confirmed whether this indirect plasma has an anti-tumor effect against CCC, and investigated whether this efficacy is selective for cancer cells. Non-thermal atmospheric pressure plasma induced apoptosis in CCC cells, while human peritoneal mesothelial cells remained viable. Non-thermal atmospheric pressure plasma exhibits selective cytotoxicity against CCC cells which are resistant to chemotherapy.

Expression of Urocortin I in Normal Tissues and Malignant Tumors

Corticotropin-releasing factor (CRF) is a key player in the hypothalamus-pituitary adrenal axis, and exerts its actions through two types of receptors: CRF type 1 and type 2 (CRF1 and CRF2) receptors. After the discovery of CRF and these receptors, a CRF-related peptide, urocortin I (UCN I), was identified. UCN I is a more potent agonist than CRF on CRF receptors, especially CRF2, which is involved in stress response. UCN I has various beneficial actions and is distributed in central and peripheral normal tissues, including the brain, heart, vascular cells, and endometrium. Recently, UCN I expression was also discovered in malignant cells, such as human glioblastoma cells, pituitary adenoma cells, hepatic carcinoma cells, gastric cancer cells, adrenocortical adenoma cells, and renal clear-cell carcinoma cells. Recent developments into the pathophysiological roles of UCN I and its related peptides will potentially lead to new anticancer therapies. Here, we review the expression of UCN I in normal and malignant cells and discuss the future of UCNs in anticancer therapy.

Combination treatment with trabectedin and irinotecan or topotecan has synergistic effects against ovarian clear cell carcinoma cells.

The objective of this study was to investigate the chemotherapeutic agents that produce the strongest synergistic effects when combined with trabectedin against ovarian clear cell carcinoma (CCC), which is regarded as an aggressive chemoresistant histological subtype. Using 4 human CCC cell lines (RMG1, RMG2, KOC7C, and HAC2), the cytotoxicities of trabectedin, SN-38, topotecan, doxorubicin, cisplatin, and paclitaxel as single agents were first assessed using the MTS assay. Then, the cytotoxicities of combination treatments involving trabectedin and 1 of the other 4 agents were evaluated by isobologram analysis to examine whether these combinations displayed synergistic, additive, or antagonistic effects. The antitumor activities of the combination treatments were also examined using cisplatin-resistant and paclitaxel-resistant CCC sublines, which were derived from the parental CCC cells by continuously exposing them to cisplatin or paclitaxel. Finally, we determined the effect of everolimus on the antitumor efficacy of trabectedin-based combination chemotherapy. Concurrent exposure to trabectedin and SN-38 or topotecan resulted in synergistic interactions in all 4 CCC cell lines. Among the tested combinations, trabectedin plus SN-38 was the most effective cytotoxic regimen. The combination of trabectedin plus SN-38 also had strong synergistic effects on both the cisplatin-resistant and paclitaxel-resistant CCC cell lines. Treatment with everolimus significantly enhanced the antitumor activity of trabectedin plus SN-38 or topotecan. Combination treatment with trabectedin and SN-38 displays the greatest cytotoxic effect against ovarian CCC. Our in vitro study provides the rationale for future clinical trials of trabectedin plus irinotecan with or without everolimus in patients with ovarian CCC in both the front-line chemotherapy setting and as a second-line treatment of recurrent CCC that had previously been treated with cisplatin or paclitaxel.

Combination of therapy with 5-fluorouracil and cisplatin with electroporation in human ovarian carcinoma model in vitro

High electric field, applied to plasma membrane, affects organization of the lipid molecules, generating transient hydrophilic electropores. The application of the cell membrane electroporation in combination with cytotoxic drugs could increase the drug transport into cells. This approach is known as electrochemotherapy (ECT). Our work shows new data concerning the influence of electrochemical reaction with cisplatin or with 5-fluorouracil (5-FU) on cancer ovarian cells resistant to standard therapy with cisplatin, in comparison to ECT effect on human primary fibroblasts. We investigated the effect of electroporation and electrochemotherapy with 5-FU and cisplatin on human ovarian clear-cell carcinoma cell line (OvBH-1) and epithelial ovarian carcinoma cell line (SKOV-3) – both resistant to cisplatin typically used in ovarian cancers. As control cells, human gingival fibroblasts (HGF's) from primary culture were used. Electropermeabilization efficiency was determined by FACS analysis with iodide propidium. Efficiency of electrochemotherapy was evaluated with viability assay. The cytotoxic effect was dependent on the electroporation parameters and on drug concentration. Electroporation alone only insignificantly decreased cells proliferation in OvBH-1 line; SKOV-3 line was more sensitive to the electrical field. Electrochemotherapy with cisplatin and 5-FU showed promising effects on both ovarian cell lines with recovery of normal cells revealed after 72hours.

X-chromosome-linked inhibitor of apoptosis as a key factor for chemoresistance in clear cell carcinoma of the ovary.

Background:X-chromosome-linked inhibitor of apoptosis (XIAP) is one of the anti-apoptotic proteins leading to chemoresistance in several cancers. The aim of this study is to evaluate the impact of XIAP expression upon ovarian clear cell carcinoma (CCC) that has a platinum-resistant phenotype.Methods:Tissue microarrays made from 90 CCC patients were analysed for immunohistochemical expression levels of XIAP, c-Met, p-Akt and Bcl-XL. In addition, CCC cell lines were evaluated whether XIAP silencing could modulate sensitivity to platinum agent in vitro.Results:High XIAP expression was observed in 30 (33%) of 90 CCC cases, and was associated with c-Met (<0.01) and Bcl-XL (<0.01) expression. Cases with high XIAP expression had lower response rate to primary platinum-based chemotherapy (10% vs 65%, P=0.02). In stages II–IV tumours, high XIAP expression was related with worse progression-free survival (PFS, P=0.02). Furthermore, high XIAP expression was identified as an independent worse prognostic factor for PFS and overall survival. Finally, downregulation of XIAP using XIAP-specific small interfering RNA increased sensitivity to cisplatin in human cancer cells derived from CCC.Conclusions:X-chromosome-linked inhibitor of apoptosis expression was correlated with chemoresistance of primary chemotherapy, and identified as a prognostic marker for CCC. X-chromosome-linked inhibitor of apoptosis could be a candidate for new therapeutic target in CCC.

Suppression of REV7 enhances cisplatin sensitivity in ovarian clear cell carcinoma cells

Human REV7 (also known as MAD2L2 and MAD2B) is involved in DNA repair, cell cycle regulation, gene transcription, and carcinogenesis. In this study, we evaluated the expression of REV7 in epithelial ovarian cancer (EOC) and analyzed the association between its expression and chemosensitivity in ovarian clear cell carcinoma (CCC) cells. Expression of REV7 in human EOC tissues was assessed by immunohistochemical staining. Expression was detected in the majority of EOCs (92.0%) with especially high levels of expression frequently observed in CCCs (73.5%) compared with that of non-CCCs (53.4%). Enhanced immunoreactivity to REV7 was associated with poor prognosis represented by reduced progression-free survival in advanced stage (stage II–IV) EOC as assessed using Kaplan–Meier curves and log–rank tests. The effects of REV7 knockdown on cell proliferation and chemosensitivity in CCC cells were also analyzed in vitro and in vivo. Knockdown of REV7 in CCC cells decreased cell proliferation without affecting cell cycle distribution. Additionally, the number of apoptotic cells and DNA damaged cells were increased after cisplatin treatment. In a nude mouse tumor xenograft model, inoculated REV7-knockdown tumors showed significantly reduced tumor volumes after cisplatin treatment compared with those of the control group. These findings indicate that depletion of REV7 enhances sensitivity to cisplatin treatment in CCC, suggesting that REV7 is a candidate molecular target in CCC management.

Epigenetic determinants of ovarian clear cell carcinoma biology.

Targeted approaches have revealed frequent epigenetic alterations in ovarian cancer, but the scope and relation of these changes to histologic subtype of disease is unclear. Genome-wide methylation and expression data for 14 clear cell carcinoma (CCC), 32 non-CCC and four corresponding normal cell lines were generated to determine how methylation profiles differ between cells of different histological derivations of ovarian cancer. Consensus clustering showed that CCC is epigenetically distinct. Inverse relationships between expression and methylation in CCC were identified, suggesting functional regulation by methylation, and included 22 hypomethylated (UM) genes and 276 hypermethylated (HM) genes. Categorical and pathway analyses indicated that the CCC-specific UM genes were involved in response to stress and many contain hepatocyte nuclear factor (HNF) 1-binding sites, while the CCC-specific HM genes included members of the estrogen receptor alpha (ERalpha) network and genes involved in tumor development. We independently validated the methylation status of 17 of these pathway-specific genes, and confirmed increased expression of HNF1 network genes and repression of ERalpha pathway genes in CCC cell lines and primary cancer tissues relative to non-CCC specimens. Treatment of three CCC cell lines with the demethylating agent Decitabine significantly induced expression for all five genes analyzed. Coordinate changes in pathway expression were confirmed using two primary ovarian cancer datasets (p < 0.0001 for both). Our results suggest that methylation regulates specific pathways and biological functions in CCC, with hypomethylation influencing the characteristic biology of the disease while hypermethylation contributes to the carcinogenic process.