Chemotherapy-resistant Ovarian Cancer Cell Research Articles

Puerarin induces platinum-resistant epithelial ovarian cancer cell apoptosis by targeting SIRT1.

ObjectivePrevious investigations indicated the anticancer activity of puerarin. The current study aimed to evaluate the effect and molecular mechanisms of puerarin in chemotherapy-resistant ovarian cancer cells.MethodsWe examined the effects of puerarin in platinum-resistant epithelial ovarian cancer cells in vitro and in vivo. We also analyzed the molecular mechanism underlying Wnt/β-catenin inhibition and sirtuin 1 (SIRT1) regulation following puerarin treatment.ResultsOur study demonstrated that puerarin effectively inhibited cell growth in vitro and in vivo by increasing apoptosis in ovarian cancer cells. More importantly, puerarin sensitized cisplatin-resistant ovarian cancer cells to chemotherapy. Puerarin treatment decreased SIRT1 expression, which attenuated the nuclear accumulation of β-catenin to inhibit Wnt/β-catenin signaling. In addition, SIRT1 overexpression diminished the effects of puerarin treatment on cisplatin-resistant ovarian cancer cells. Further analysis supported SIRT1/β-catenin expression as a candidate biomarker for the disease progression of epithelial ovarian cancer.ConclusionsPuerarin increased the apoptosis of platinum-resistant ovarian cancer cells. The mechanism is partly related to the downregulation of SIRT1 and subsequent inhibition of Wnt/β-catenin signaling.

Abstract B44: Protein interactions involving LARP1 in chemotherapy resistant ovarian cancer cells

Abstract Ovarian cancer (OC) is the most lethal gynecological malignancy accounting for 152,000 deaths annually worldwide. Due to the lack of a validated diagnostic tool, patients usually present with disseminated disease and the majority will develop resistance to platinum chemotherapy. Resensitizing OC to cisplatin-based chemotherapy is an unmet clinical need. The RNA-binding protein LARP1 is highly expressed in OC and is a post-transcriptional regulator of cell survival and tumorigenesis. LARP1 depletion using RNA interference (RNAi) restores platinum sensitivity in cisplatin-resistant OC cell lines and shows a synergistic anti-tumour effect with cisplatin. We explored the effect of this resensitization on the efficiency of protein translation using the Surface Sensing of Translation (SUnSET) method. Whilst cisplatin-induced genotoxic stress attenuated de novo protein synthesis in platinum sensitive OVCAR3 cells, this was not observed in platinum-resistant OVCAR8 cells where residual protein synthesis was maintained. However this residual translation was completely abolished after RNAi to LARP1 suggesting that, in the OVCAR8 cells, LARP1 maintains protein synthesis during genotoxic stress. Although LARP1 is known to bind Poly A binding protein (PABP) we questioned whether other interacting partners might also regulate this response. Using immunoprecipitation followed by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) in OVCAR3 and OVCAR8 cells before and after cisplatin treatment, we identified PABPC1, PABPC4 and YB-1 as strong interactors with LARP1. By western blotting, Duolink Proximity Ligation Assay and Immunofluorescence we then confirmed these candidates as being complexed with LARP1. We are currently elucidating the functional role of these interactions on the LARP1-mediated genotoxic stress response with the overall aim of identifying therapeutic strategies to overcome platinum-resistance in ovarian cancer. Citation Format: Chara Stavraka, Manuela Mura, Roman Fischer, Marie-Laetitia Thezenas, Sadaf Ghaem Maghami, James Chettle, Laki Buluwela, Benedikt Kessler, Sarah P. Blagden. Protein interactions involving LARP1 in chemotherapy resistant ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B44.

Dual-Targeting Nanoparticles for In Vivo Delivery of Suicide Genes to Chemotherapy-Resistant Ovarian Cancer Cells.

Ovarian cancer is the most lethal gynecologic cancer. Claudin-3 and -4, the receptors for Clostridium perfringens enterotoxin (CPE), are overexpressed in more than 70% of these tumors. Here, we synthesized and characterized poly(lactic-co-glycolic-acid) (PLGA) nanoparticles (NPs) modified with the carboxy-terminal-binding domain of CPE (c-CPE-NP) for the delivery of suicide gene therapy to chemotherapy-resistant ovarian cancer cells. As a therapeutic payload, we generated a plasmid encoding for the diphtheria toxin subunit-A (DT-A) under the transcriptional control of the p16 promoter, a gene highly differentially expressed in ovarian cancer cells. Flow cytometry and immunofluorescence demonstrated that c-CPE-NPs encapsulating the cytomegalovirus (CMV) GFP plasmid (CMV GFP c-CPE-NP) were significantly more efficient than control NPs modified with a scrambled peptide (CMV GFP scr-NP) in transfecting primary chemotherapy-resistant ovarian tumor cell lines in vitro (P = 0.03). Importantly, c-CPE-NPs encapsulating the p16 DT-A vector (p16 DT-A c-CPE-NP) were significantly more effective than control p16 DT-A scr-NP in inducing ovarian cancer cell death in vitro (% cytotoxicity: mean ± SD = 32.9 ± 0.15 and 7.45 ± 7.93, respectively, P = 0.03). In vivo biodistribution studies demonstrated efficient transfection of tumor cells within 12 hours after intraperitoneal injection of CMV GFP c-CPE-NP in mice harboring chemotherapy-resistant ovarian cancer xenografts. Finally, multiple intraperitoneal injections of p16 DT-A c-CPE-NP resulted in a significant inhibition of tumor growth compared with control NP in chemotherapy-resistant tumor-bearing mice (P = 0.041). p16 DT-A c-CPE-NP may represent a novel dual-targeting therapeutic approach for the selective delivery of gene therapy to chemotherapy-resistant ovarian cancer cells. Mol Cancer Ther; 16(2); 323-33. ©2016 AACR.

MiR-497 decreases cisplatin resistance in ovarian cancer cells by targeting mTOR/P70S6K1.

The mechanism of cisplatin resistance in ovarian cancer is not clearly understood. In the present investigation, we found that the expression levels of miR-497 were reduced in chemotherapy-resistant ovarian cancer cells and tumor tissues due to hypermethylation of miR-497 promoter. Low miR-497 expression levels were associated with chemo-resistant phonotype of ovarian cancer. By analyzing the expression levels of miR-497, mTOR and p70S6K1 in a clinical gene-expression array dataset, we found that mTOR and p70S6K1, two proteins correlated to chemotherapy-resistance in multiple types of human cancers, were inversely correlated with miR-497 levels in ovarian cancer tissues. By using an orthotopic ovarian tumor model and a Tet-On inducible miR-497 expression system, our results demonstrated that overexpression of miR-497 sensitizes the resistant ovarian tumor to cisplatin treatment. Therefore, we suggest that miR-497 might be used as a therapeutic supplement to increase ovarian cancer treatment response to cisplatin.

Solitomab, an EpCAM/CD3 bispecific antibody (BITE), is highly active against primary chemotherapy resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo

Solitomab, an EpCAM/CD3 bispecific antibody (BITE), is highly active against primary chemotherapy resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo

Solitomab, an epithelial cell adhesion molecule/CD3 bispecific antibody (BiTE), is highly active against primary chemotherapy-resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo.

Solitomab is a novel, bispecific, single-chain antibody that targets epithelial cell adhesion molecule (EpCAM) on tumor cells and also contains a cluster of differentiation 3 (CD3) (T-cell coreceptor) binding region. The authors evaluated the in vitro activity of solitomab against primary chemotherapy-resistant epithelial ovarian carcinoma cell lines as well as malignant cells in ascites. EpCAM expression was evaluated by flow cytometry in 5 primary ovarian cancer cell lines and in 42 fresh ovarian tumor cell cultures in ascites from patients with mainly advanced or recurrent, chemotherapy-resistant disease. The potential activity of solitomab against EpCAM-positive tumor cells was evaluated by flow cytometry, proliferation, and 4-hour chromium-release, cell-mediated cytotoxicity assays. EpCAM expression was detected by flow cytometry in approximately 80% of the fresh ovarian tumors and primary ovarian tumor cell lines tested. EpCAM-positive, chemotherapy-resistant cell lines were identified as resistant to natural killer cell-mediated or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean±standard error of the mean, 3.6%±0.7% of cells killed after incubation of EpCAM-positive cell lines with control bispecific antibody). In contrast, after incubation with solitomab, EpCAM-positive, chemotherapy-resistant cells became highly sensitive to T-cell cytotoxicity (mean±standard error of the mean, 28.2%±2.05% of cells killed; P<.0001) after exposure to peripheral blood lymphocytes. Ex vivo incubation of autologous tumor-associated lymphocytes with EpCAM-expressing malignant cells in ascites with solitomab resulted in a significant increase in T-cell activation markers and a reduction in the number of viable ovarian tumor cells in ascites (P<.001). Solitomab may represent a novel, potentially effective agent for the treatment of chemotherapy-resistant ovarian cancers that overexpress EpCAM.

Solitomab, an EpCAM/CD3 bispecific antibody (BiTE®), is highly active against primary chemotherapy-resistant ovarian cancer cell lines in vitro and fresh tumor cells ex vivo

Objectives: Chemotherapy-resistant ovarian cancer remains an incurable disease. Solitomab is a novel bispecific single-chain antibody that targets the epithelial antigen EpCAM on tumor cells and also contains a CD3-binding region. We evaluated the in vitro activity of solitomab against multiple primary chemotherapy-resistant epithelial ovarian carcinoma cell lines and malignant tumor cells in ascitic fluid.

Abstract 732: Iron oxide nanoparticles functionalized with the Clostridium Perfringens Enterotoxin carboxi-terminal fragment peptide: A novel diagnostic and therapeutic approach to specifically target ovarian cancer.

Abstract Ovarian carcinoma remains the cancer with the highest mortality among gynecological tumors. Development of novel strategies for the diagnosis and treatment of chemotherapy resistant ovarian disease remains a high priority. Magnetic iron oxide nanoparticles (NP) consist of an iron oxide core and hydrophilic coating. NP can establish magnetic field gradients that alter proton relaxation properties, thus providing an excellent source of contrast for magnetic resonance imaging (MRI). Moreover, since they can be heated in alternating magnetic fields (AMF), they may also provide an effective form of hyperthermia for cancer therapy. Unfortunately, currently available iron oxide NP, although potentially able to allow MRI detection of single cells in vivo, are not tumor specific. Our group has recently analyzed the genetic fingerprints of ovarian cancer identifying extremely high expression of the genes encoding the proteins claudin-3 and -4, the epithelial receptors for Clostridium perfringens enterotoxin (CPE). Since the C-terminus domain (C-CPE) of the toxin is sufficient for CPE binding and it is not toxic to cells, we used iron oxide NP complexed to C-CPE peptide to target chemotherapy resistant ovarian cancer cells overexpressing claudin-3 and -4. We report that NP functionalized by the fluorescent C-CPE peptide effectively bind and rapidly internalize into ovarian cancer cells using flow cytometry,confocal microscopy and Prussian blue staining. A 5-fold increase in iron cellular uptake after incubation of tumor cells with C-CPE NP was consistently detected when compared to tumor cells exposed to non specific control NP [i.e., iron concentration of (mean ± STDV) 20.3 ± 0.5 vs 4.1 ± 1.3 nmoles/5*10 cells respectively; p&amp;lt;0.01]. Importantly, only tumor cells exposed to C-CPE NP were detectable in MRI in vitro experiments using a Bruker 4.7Tmagnet. Finally, to assess the potential of C-CPE NP to be used for hyperthermia therapy against chemotherapy resistant ovarian cancer, we characterized the magnetic heating capacity of C-CPE NP using an induction heating system (Ambrell, Easy Heat). Our results show a temperature increase of 7.85 ± 0.6 °C (mean ± STDV) when C-CPE NP were subjected to an AMF of 343 kHz of frequency and 2 KW of power for 10 minutes. Our data clearly suggest that C-CPE NP may represent a novel theranostic tool to specifically target chemotherapy resistant ovarian cancer. Citation Format: Emiliano Cocco, Ileana Bortolomai, Stefania Bellone, Sara Gasparrini, Diana English, Erik Shapiro, Sergio Pecorelli, Dan-Arin Silasi, Masoud Azodi, Elena Ratner, Thomas Rutherford, Peter Schwartz, Alessandro Santin. Iron oxide nanoparticles functionalized with the Clostridium Perfringens Enterotoxin carboxi-terminal fragment peptide: A novel diagnostic and therapeutic approach to specifically target ovarian cancer. [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 732. doi:10.1158/1538-7445.AM2013-732

Smoothened Antagonists Reverse Taxane Resistance in Ovarian Cancer

The hedgehog pathway has been implicated in the formation and maintenance of a variety of malignancies, including ovarian cancer; however, it is unknown whether hedgehog signaling is involved in ovarian cancer chemoresistance. The goal of this study was to determine the effects of antagonizing the hedgehog receptor, Smoothened (Smo), on chemotherapy response in ovarian cancer. Expression of hedgehog pathway members was assessed in three pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using quantitative PCR and Western blot analysis. Cell lines were exposed to increasing concentrations of two different Smo antagonists (cyclopamine, LDE225) alone and in combination with carboplatin or paclitaxel. Selective knockdown of Smo, Gli1, or Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay. A2780cp20 and SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel, or combination therapy. Chemoresistant cell lines showed higher expression (>2-fold, P < 0.05) of hedgehog signaling components compared with their respective parental lines. Smo antagonists sensitized chemotherapy-resistant cell lines to paclitaxel, but not to carboplatin. LDE225 treatment also increased sensitivity of ALDH-positive cells to paclitaxel. A2780cp20 and SKOV3TRip2 xenografts treated with combined LDE225 and paclitaxel had significantly less tumor burden than those treated with vehicle or either agent alone. Increased taxane sensitivity seems to be mediated by a decrease in P-glycoprotein (MDR1) expression. Selective knockdown of Smo, Gli1, or Gli2 all increased taxane sensitivity. Smo antagonists reverse taxane resistance in chemoresistant ovarian cancer models, suggesting combined anti-hedgehog and chemotherapies could provide a useful therapeutic strategy for ovarian cancer.

Mammaglobin B (SCGB2A1)-specific CD8 + cytotoxic T lymphocytes (CTL) are highly effective in killing autologous chemotherapy resistant ovarian cancer cells: Implications for SCGB2A1 dendritic cell-based therapeutic vaccines

Objective: Objective: To evaluate the potential of SCGB2A1, a top differentially expressed gene in various histological types of epithelial ovarian carcinomas, as a novel tumor antigen for ovarian cancer immunotherapy.

Combination of a MDR1-targeted replicative adenovirus and chemotherapy for the therapy of pretreated ovarian cancer

Targeted oncolytic adenoviruses capable of replication selectively in cancer cells are an appealing approach for the treatment of various cancer types refractory to conventional therapies. The aim of this study was to evaluate the effect of Ad5/3MDR1E1, a multidrug resistance gene 1 (MDR1)-targeted fiber-modified replication-competent adenovirus for the therapy of platinum-pretreated ovarian cancer in combination with cytostatic agents. MDR1-specific tumor cell killing of Ad5/3MDR1E1 was systematically evaluated in chemotherapy naïve and pretreated ovarian cancer cells in vitro. Combinations of Ad5/3MDR1E1 and cytostatic agents were studied in vivo and in vitro. An in vivo hepatotoxicity model was used to evaluate liver toxicity. We demonstrate efficient oncolysis of Ad5/3MDR1E1 in chemotherapy-resistant ovarian cancer cells as well as therapeutic efficacy in an orthotopic mouse model. Further, combining Ad5/3MDR1E1 with paclitaxel resulted in greater therapeutic benefit than either agent alone. These preclinical data suggest that a fiber-modified adenovirus vector under the control of the MDR1 promoter represents a promising treatment strategy for platinum-pretreated ovarian cancer as a single agent or in combination with conventional anticancer drugs.

Treatment of chemotherapy resistant ovarian cancer with a MDR1 targeted oncolytic adenovirus

Objective Multidrug resistance gene 1 (MDR1) mediated resistance to chemotherapeutic agents is a major obstacle for the therapy of various cancer types. The use of conditionally replicating adenoviruses (CRAds) is dependent on molecular differences between tumor cells and non tumor cells. Transcriptional targeting of CRAd replication is an effective way to control replication regulation. The aim of this study was to evaluate the effect of a MDR1 targeted fiber-modified CRAd against chemotherapy resistant ovarian cancer. Methods MDR1 expression was evaluated in chemotherapy naïve and pretreated ovarian cancer cells and various control cells. We constructed 2 variants of a fiber-modified CRAd, Ad5/3MDR1E1 and Ad5/3MDR1E1∆24 containing the MDR1 promoter to control viral replication via the E1A gene. The MDR promoter activity and cell killing efficacy were evaluated in vitro. Orthotopic murine models of peritoneally disseminated ovarian cancer were utilized to evaluate the preclinical efficacy of MDR targeted CRAds in vivo. To evaluate the liver toxicity of MDR1 targeted CRAds, we compared Ad5/3MDR1E1 with Ad5/3∆24, a CRAd that replicates in cancer cells inactive in the Rb/p16 pathway by use of an in vivo hepatotoxicity model. Results We demonstrate efficient oncolysis of Ad5/3MDR1E1 in both chemotherapy resistant ovarian cancer cell lines and in primary tumor cells from pretreated patients as well as therapeutic efficacy in an orthotopic mouse model. Ad5/3MDR1E1 demonstrated significantly decreased liver toxicity compared to other 5/3-fiber modified control vectors examined. Conclusions In summary, Ad5/3MDR1E1 is an efficient and safe gene therapy approach for specific targeting of chemotherapy resistant cancer cells.

Tissue factor expression in ovarian cancer: implications for immunotherapy with hI-con1, a factor VII-IgGFc chimeric protein targeting tissue factor

We evaluated the expression of tissue factor (TF) in ovarian cancer (EOC) and the potential of hI-con1, an antibody-like molecule targeting TF, as a novel form of therapy against chemotherapy-resistant ovarian disease. We studied the expression of TF in 88 EOC by immunohistochemistry (IHC) and real-time-PCR (qRT-PCR) and the levels of membrane-bound-complement-regulatory-proteins CD46, CD55 and CD59 in primary EOC cell lines by flow-cytometry. Sensitivity to hI-con1-dependent-cell-mediated-cytotoxicity (IDCC), complement-dependent-cell-cytotoxicity and inhibition of IDCC by γ-immunoglobulin were evaluated in 5-h (51)chromium-release-assays. Cytoplasmic and/or membrane TF expression was observed in 24 out of 25 (96%) of the EOC samples tested by IHC, but not in normal ovarian-tissue. EOC with clear cell histology significantly overexpress TF when compared to serous, endometrioid, or undifferentiated tumors by qRT-PCR. With a single exception, all primary EOC that overexpressed TF demonstrated high levels of CD46, CD55 and CD59 and regardless of their histology or resistance to chemotherapy, were highly sensitive to IDCC. The effect of complement and physiologic doses of γ-immunoglobulin on IDCC in ovarian cancer cell lines overexpressing TF was tumor specific and related to the overexpression of CD59 on tumor cells. Small-interfering-RNA-mediated knockdown of CD59 expression in ovarian tumors significantly increased hI-con1-mediated cytotoxic activity in vitro. Finally, low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (P<0.01). hI-con1 molecule induces strong cytotoxicity against primary chemotherapy-resistant ovarian cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of ovarian tumors refractory to standard treatment modalities.

Abstract 1728: Targeting hedgehog reverses taxane resistance by Gli-dependent and independent mechanisms in ovarian cancer

Abstract Objective: Recent studies have implicated hedgehog signaling in the formation and continued growth of a variety of malignancies, including ovarian cancer. Several inhibitors of the hedgehog pathway have been identified that block the activity of the Smoothened (Smo) receptor. The goal of this study was to determine the in vitro and in vivo effects of Smo antagonists alone and in combination with chemotherapy in ovarian cancer. Methods: Expression of hedgehog signaling components (Smo, Gli1 and Gli2) was assessed in 3 pairs of parental and chemotherapy-resistant ovarian cancer cell lines (A2780ip2/A2780cp20, SKOV3ip1/SKOV3TRip2, HeyA8/HeyA8MDR) using Western blot and qPCR. Cell lines were exposed to increasing concentrations of two different Smo antagonists (Cyclopamine, LDE225) alone and in combination with carboplatin, paclitaxel, adriamycin, and topotecan. Selective knockdown of Smo, Gli1 and Gli2 was achieved using siRNA constructs. Cell viability was assessed by MTT assay and PARP cleavage was used as an indicator of apoptosis. SKOV3TRip2 orthotopic xenografts were treated with vehicle, LDE225, paclitaxel or combination therapy for 5 weeks. Tumor weight for each treatment group was measured and compared using student's t-test. Results: Expression of Smo and Gli1 was high in A2780ip2/A2780cp20, moderate in SKOV3ip2/SKOV3TRip2 and low/absent in HeyA8/HeyA8MDR. Gli2 was high in SKOV3ip2/SKOV3TRip2, moderate in A2780ip2/A2780cp20 and low in HeyA8/HeyA8MDR. Response to cyclopamine and LDE225 varied among the cell lines examined with IC50s ranging from 7.5 to &amp;gt;20 µM. Both agents sensitized chemotherapy-resistant cell lines to paclitaxel (5- to 26-fold, including Smo[low]/Gli1[neg] HeyA8MDR), but not to carboplatin, adriamycin, or topotecan. Selective knockdown of Gli1 and Gli2 resulted in taxane sensitization only in Gli1/2-high A2780cp20 cells (2- to 8-fold). A decrease in acetyl-α-tubulin confirmed microtubule-specific effects of Smo targeting, supporting the taxane specificity of this effect. In vivo, SKOV3TRip2 xenografts treated with LDE225 or paclitaxel alone had slightly less tumor burden than the control group (reduced by 28.1%, p=0.42 and 32.0%, p=0.40, respectively). Those treated with combined LDE225 and paclitaxel, however, had significantly less tumor burden than those treated with vehicle (70.5% reduction, p=0.015). Conclusions: Targeting the hedgehog pathway decreases cell viability and increases taxane sensitivity in taxane-resistant ovarian cancer models. Interestingly, these effects were noted even in cells with little constitutive hedgehog activity. This suggests both Gli-dependent and -independent mechanisms contribute to taxane resistance, expanding the potential use of hedgehog inhibitors to all taxane-resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1728. doi:10.1158/1538-7445.AM2011-1728

High-grade, chemotherapy-resistant primary ovarian carcinoma cell lines overexpress human trophoblast cell-surface marker (Trop-2) and are highly sensitive to immunotherapy with hRS7, a humanized monoclonal anti-Trop-2 antibody

Objective. We evaluated the expression of human trophoblast cell-surface marker (Trop-2) and the potential of hRS7, a humanized monoclonal anti-Trop-2 antibody, as a therapeutic agent against chemotherapy-resistant ovarian disease. Methods. Trop-2 expression was evaluated by immunohistochemistry (IHC) in 50 ovarian serous papillary carcinoma specimens. Trop-2 expression was also evaluated by real-time PCR (qRT-PCR) and flow cytometry in a total of 6 primary ovarian cancer cell lines derived from patients with chemotherapy-resistant disease. Sensitivity to hRS7 antibody-dependent cellular cytotoxicity (ADCC) was tested in standard 5-hour 51Cr-release assays. The effect of serum and interleukin-2 (IL-2) on hRS7-mediated ADCC was also studied. Results. Trop-2 expression was found in 41 of 50 (82%) tumor tissues tested by IHC. 83% (5 of 6) of the ovarian cancer cell lines tested by qRT-PCR and flow cytometry demonstrated high Trop-2 expression. All primary ovarian cancer cell lines expressing Trop-2 were highly sensitive to hRS7-mediated ADCC in vitro (range of killing: 19.3% to 40.8%) ( p < 0.001). Negligible cytotoxicity against chemotherapy-resistant ovarian cancers was seen in the absence of hRS7 or in the presence of rituximab control antibody (range of killing: 1.1% to 8.9%). Human serum did not significantly inhibit hRS7-mediated cytotoxicity while incubation with IL-2 in addition to hRS7 further increased the cytotoxic activity ( p = 0.04). Conclusions. Trop-2 is highly expressed in chemotherapy-resistant ovarian cancer cell lines at mRNA and protein levels. Primary ovarian carcinoma cell lines are highly sensitive to hRS7-mediated cytotoxicity in vitro. hRS7 may represent a novel therapeutic agent for the treatment of high-grade, chemotherapy-resistant ovarian cancer.

Clostridium perfringens enterotoxin carboxy-terminal fragment is a novel tumor-homing peptide for human ovarian cancer

BackgroundDevelopment of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority. Using high-throughput technologies to analyze genetic fingerprints of ovarian cancer, we have discovered extremely high expression of the genes encoding the proteins claudin-3 and claudin-4.MethodsBecause claudin-3 and -4 are the epithelial receptors for Clostridium perfringens enterotoxin (CPE), and are sufficient to mediate CPE binding, in this study we evaluated the in vitro and in vivo bioactivity of the carboxy-terminal fragment of CPE (i.e., CPE290-319 binding peptide) as a carrier for tumor imaging agents and intracellular delivery of therapeutic drugs. Claudin-3 and -4 expression was examined with rt-PCR and flow cytometry in multiple primary ovarian carcinoma cell lines. Cell binding assays were used to assess the accuracy and specificity of the CPE peptide in vitro against primary chemotherapy-resistant ovarian carcinoma cell lines. Confocal microscopy and biodistribution assays were performed to evaluate the localization and uptake of the FITC-conjugated CPE peptide in established tumor tissue.ResultsUsing a FITC-conjugated CPE peptide we show specific in vitro and in vivo binding to multiple primary chemotherapy resistant ovarian cancer cell lines. Bio-distribution studies in SCID mice harboring clinically relevant animal models of chemotherapy resistant ovarian carcinoma showed higher uptake of the peptide in tumor cells than in normal organs. Imunofluorescence was detectable within discrete accumulations (i.e., tumor spheroids) or even single chemotherapy resistant ovarian cancer cells floating in the ascites of xenografted animals while a time-dependent internalization of the FITC-conjugated CPE peptide was consistently noted in chemotherapy-resistant ovarian tumor cells by confocal microscopy.ConclusionsBased on the high levels of claudin-3 and -4 expression in chemotherapy-resistant ovarian cancer and other highly aggressive human epithelial tumors including breast, prostate and pancreatic cancers, CPE peptide holds promise as a lead peptide for the development of new diagnostic tracers or alternative anticancer agents.

EpCAM-specific immunotherapy with human monoclonal antibody adecatumumab (MT201) in chemotherapy-resistant ovarian cancer cell lines.

e15524 Background: To evaluate the expression of epithelial cell adhesion molecule (EpCAM) and to determine the cytotoxic potential of MT201 (adecatumumab), a fully human recombinant IgG1 anti-EpCAM antibody, against multiple primary chemotherapy- resistant ovarian cancer cell lines in vitro. Methods: EpCAM expression was evaluated by real time-PCR and flow cytometry in a total of seven primary ovarian cancer cell lines established from patients who showed disease progression on multiple chemotherapeutic regimens. Sensitivity to MT201 antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) was tested in these primary ovarian cancer cell lines expressing different levels of EpCAM in standard 5-hour 51Cr assays. To investigate the effect of interleukin-2 (IL-2) on MT201-mediated ADCC, 5-hour 51Cr assays were also conducted after stimulation with low doses of IL-2. Results: Five papillary-serous and two clear cell ovarian carcinoma cell lines were evaluated. High EpCAM surface expression by flow cytometry and high mRNA expression by real time-PCR was detected in 71% of primary ovarian cancer cell lines (five out of seven cell lines). While these cell lines were highly resistant to CDC and natural killer dependent cytotoxicity in vitro (range of killing 0% to 7%), EpCAM-positive cell lines showed very high sensitivity to MT201-mediated ADCC (range of killing 27% to 66%). Incubation with IL-2 in addition to MT201 significantly increased the cytotoxic activity against EpCAM-positive ovarian cancer cell lines (p < 0.0001). Conclusions: EpCAM is highly expressed in primary chemotherapy- resistant ovarian carcinoma cell lines, and these chemotherapy-resistant tumors are highly sensitive to MT201-mediated cytotoxicity in vitro. MT201 may represent a novel, potentially highly effective treatment option for patients harboring chemotherapy-resistant ovarian carcinoma. No significant financial relationships to disclose.

Untersuchungen zur TRAIL-induzierten Apoptose von Ovarialkarzinomzelllinien mit selektiver Zytostatikaresistenz

Chemotherapy resistance remains a key problem in the treatment of advanced ovarian cancer. Usage of various drug combinations has been successful to improve therapy response and relapse-free survival. Nonetheless, the majority of ovarian cancers relapse, consecutively developing drug resistance. The combination of TRAIL (TNF-Related Apoptosis Inducing Ligand) and various cytostatic drugs such as platin derivates and taxanes has shown synergistic effects in tissue culture models when cancer cell lines of different origins were treated. Our study was performed to evaluate whether TRAIL can cause apoptosis and/or growth inhibition of chemotherapy-resistant ovarian cancer cell lines. To develop ovarian cancer cell lines with selective drug resistance, the parental cell line HEY was treated with increasing drug concentrations of cisplatin, etoposide, docetaxel, and paclitaxel, respectively. Growth inhibition was evaluated with the formazan-based WST-1 assay (Roche); apoptosis induction was evaluated with the Cell Death Detection ELISA (Roche). All resistant cell lines showed growth inhibition and apoptosis when treated with TRAIL. Apart from the cisplatin-resistant cell line, which responded almost similarly compared to the parental cell line, the TRAIL response of all other resistant cell lines was significantly weaker than of the parental cell line. Cisplatin-resistant cells showed the most, docetaxel-resistant cells the least response. TRAIL-resistant cells showed less growth inhibition as well as less apoptosis than parental cells when treated with different cytostatic drugs. The synergistic effects of cytostatic drugs and TRAIL and the responsse of chemoresistant ovarian cancer cells to TRAIL suggest TRAIL can be a therapeutic option for the treatment of advanced ovarian cancer.