Publications by authors named "Laurent Brard"

50 Publications

Assessment of acyl-CoA cholesterol acyltransferase (ACAT-1) role in ovarian cancer progression-An in vitro study.

PLoS One 2020 24;15(1):e0228024. Epub 2020 Jan 24.

Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Southern Illinois University School of Medicine, Springfield, IL, United States America.

Abnormal accumulation of acyl-CoA cholesterol acyltransferase-1 (ACAT-1) mediated cholesterol ester has been shown to contribute to cancer progression in various cancers including leukemia, glioma, breast, pancreatic and prostate cancers. However, the significance of ACAT-1 and cholesterol esters (CE) is relatively understudied in ovarian cancer. In this in vitro study, we assessed the expression and contribution of ACAT-1 in ovarian cancer progression. We observed a significant increase in the expression of ACAT-1 and CE levels in a panel of ovarian cancer cell lines (OC-314, SKOV-3 and IGROV-1) compared to primary ovarian epithelial cells (normal controls). To confirm the tumor promoting capacity of ACAT-1, we inhibited ACAT-1 expression and activity by treating our cell lines with an ACAT inhibitor, avasimibe, or by stable transfection with ACAT-1 specific short hairpin RNA (shRNA). We observed significant suppression of cell proliferation, migration and invasion in ACAT-1 knockdown ovarian cancer cell lines compared to their respective controls (cell lines transfected with scrambled shRNA). ACAT-1 inhibition enhanced apoptosis with a concurrent increase in caspases 3/7 activity and decreased mitochondrial membrane potential. Increased generation of reactive oxygen species (ROS) coupled with increased expression of p53 may be the mechanism(s) underlying pro-apoptotic action of ACAT-1 inhibition. Additionally, ACAT-1 inhibited ovarian cancer cell lines displayed enhanced chemosensitivity to cisplatin treatment. These results suggest ACAT-1 may be a potential new target for the treatment of ovarian cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0228024PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980601PMC
April 2020

Assessment of peritoneal microbial features and tumor marker levels as potential diagnostic tools for ovarian cancer.

PLoS One 2020 9;15(1):e0227707. Epub 2020 Jan 9.

Department of Medical Microbiology, Immunology and Cell Biology, SIU School of Medicine, Springfield, Illinois, United States of America.

Epithelial ovarian cancer (OC) is the most deadly cancer of the female reproductive system. To date, there is no effective screening method for early detection of OC and current diagnostic armamentarium may include sonographic grading of the tumor and analyzing serum levels of tumor markers, Cancer Antigen 125 (CA-125) and Human epididymis protein 4 (HE4). Microorganisms (bacterial, archaeal, and fungal cells) residing in mucosal tissues including the gastrointestinal and urogenital tracts can be altered by different disease states, and these shifts in microbial dynamics may help to diagnose disease states. We hypothesized that the peritoneal microbial environment was altered in patients with OC and that inclusion of selected peritoneal microbial features with current clinical features into prediction analyses will improve detection accuracy of patients with OC. Blood and peritoneal fluid were collected from consented patients that had sonography confirmed adnexal masses and were being seen at SIU School of Medicine Simmons Cancer Institute. Blood was processed and serum HE4 and CA-125 were measured. Peritoneal fluid was collected at the time of surgery and processed for Next Generation Sequencing (NGS) using 16S V4 exon bacterial primers and bioinformatics analyses. We found that patients with OC had a unique peritoneal microbial profile compared to patients with a benign mass. Using ensemble modeling and machine learning pathways, we identified 18 microbial features that were highly specific to OC pathology. Prediction analyses confirmed that inclusion of microbial features with serum tumor marker levels and control features (patient age and BMI) improved diagnostic accuracy compared to currently used models. We conclude that OC pathogenesis alters the peritoneal microbial environment and that these unique microbial features are important for accurate diagnosis of OC. Our study warrants further analyses of the importance of microbial features in regards to oncological diagnostics and possible prognostic and interventional medicine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227707PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952086PMC
April 2020

Utility and Generalizability of Multistate, Population-Based Cancer Registry Data for Rural Cancer Surveillance Research in the United States.

Cancer Epidemiol Biomarkers Prev 2018 11 21;27(11):1252-1260. Epub 2018 Mar 21.

Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Southern Illinois University School of Medicine, Springfield, Illinois.

More than 46 million Americans live in rural areas, but rural populations remain relatively understudied in cancer disparities research. However, several analyses of multistate cancer registry data that describe the rural cancer incidence burden have been recently published. In light of this, our article aims to characterize the utility and generalizability of multistate, population-based cancer registry datasets for rural cancer surveillance research. First, we describe the accessibility, geographic coverage, available variables, and strengths and weaknesses of five data sources. Second, we evaluate two of these data sources-the North American Association of Central Cancer Registries (NAACCR) public use dataset (93% population coverage) and the Surveillance Epidemiology and End Results (SEER) 18 dataset (28% population coverage)-on their characterization of rural-urban cancer incidence rates and sociodemographic representation. The five data sources varied in geographic coverage and extent of available variables. SEER 18's cancer rates sociodemographic representation differed from the more geographically representative NAACCR data. We suggest that SEER increase its geographic coverage to improve their generalizability and to take advantage of their utility to assess disparities along the cancer control continuum. We also suggest that non-SEER data sources be utilized more frequently to capitalize on their extensive geographic coverage.
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http://dx.doi.org/10.1158/1055-9965.EPI-17-1087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150847PMC
November 2018

Rural-urban differences in surgical treatment, regional lymph node examination, and survival in endometrial cancer patients.

Cancer Causes Control 2018 02 27;29(2):221-232. Epub 2017 Dec 27.

Department of Obstetrics and Gynecology, Southern Illinois University School of Medicine, Springfield, IL, USA.

Purpose: Endometrial cancer (EC) is the most common gynecological malignancy and one of few cancers with an increasing US mortality rate. Rural patients may have less access to specialty care affecting their receipt of surgery and adequate lymphadenectomy (AL). We sought to assess rural-urban differences in EC surgery, lymphadenectomy, and survival.

Methods: We analyzed data from the Surveillance Epidemiology and End Results database on EC patients (2004-2013). We performed univariate analyses to compare rural and urban patients on demographic and clinical characteristics and receipt of nodal examination and AL. We assessed rural-urban differences in trends of receipt of AL, performed logistic regression to evaluate differences in receipt of surgery, nodal examination, and AL, and performed survival analysis.

Results: Rural patients were less likely to have any lymph nodes removed, had a smaller median number removed, and a smaller proportion had AL. Even after controlling for established risk factors, rural patients had lower odds of lymph node examination and adequate AL than urban patients and also had poorer survival.

Conclusions: Future research should continue to assess the association between access to care and disparities in surgical care and the effect of these disparities on survival.
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http://dx.doi.org/10.1007/s10552-017-0998-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311991PMC
February 2018

Evaluation of the cytotoxicity of the Bithionol-paclitaxel combination in a panel of human ovarian cancer cell lines.

PLoS One 2017 20;12(9):e0185111. Epub 2017 Sep 20.

Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Southern Illinois University School of Medicine, Springfield, IL, United States of America.

Previously, Bithionol (BT) was shown to enhance the chemosensitivity of ovarian cancer cell lines to cisplatin treatment. In the present study, we focused on the anti-tumor potential of the BT-paclitaxel combination when added to a panel of ovarian cancer cell lines. This in vitro study aimed to 1) determine the optimum schedule for combination of BT and paclitaxel and 2) assess the nature and mechanism(s) underlying BT-paclitaxel interactions. The cytotoxic effects of both drugs either alone or in combination were assessed by presto-blue cell viability assay using six human ovarian cancer cell lines. Inhibitory concentrations to achieve 50% cell death (IC50) were determined for BT and paclitaxel in each cell line. Changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27 were determined via immunoblot. Luminescent and colorimetric assays were used to determine caspases 3/7 and autotaxin (ATX) activity. Cellular reactive oxygen species (ROS) were measured by flow cytometry. Our results show that the efficacy of the BT-paclitaxel combination depends upon the concentrations and sequence of addition of paclitaxel and BT. Pretreatment with BT followed by paclitaxel resulted in antagonistic interactions whereas synergistic interactions were observed when both drugs were added simultaneously or when cells were pretreated with paclitaxel followed by BT. Synergistic interactions between BT and paclitaxel were attributed to increased ROS generation and enhanced apoptosis. Decreased expression of pro-survival factors (XIAP, bcl-2, bcl-xL) and increased expression of pro-apoptotic factors (caspases 3/7, PARP cleavage) was observed. Additionally, increased expression of key cell cycle regulators p21 and p27 was observed. These results show that BT and paclitaxel interacted synergistically at most drug ratios which, however, was highly dependent on the sequence of the addition of drugs. Our results suggest that BT-paclitaxel combination therapy may be effective in sensitizing ovarian cancer cells to paclitaxel treatment, thus mitigating some of the toxic effects associated with high doses of paclitaxel.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185111PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5607185PMC
October 2017

Rural-Urban Differences in Cancer Incidence and Trends in the United States.

Cancer Epidemiol Biomarkers Prev 2018 11 27;27(11):1265-1274. Epub 2017 Jul 27.

Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Southern Illinois University School of Medicine, Springfield, Illinois.

Cancer incidence and mortality rates in the United States are declining, but this decrease may not be observed in rural areas where residents are more likely to live in poverty, smoke, and forego cancer screening. However, there is limited research exploring national rural-urban differences in cancer incidence and trends. We analyzed data from the North American Association of Central Cancer Registries' public use dataset, which includes population-based cancer incidence data from 46 states. We calculated age-adjusted incidence rates, rate ratios, and annual percentage change (APC) for: all cancers combined, selected individual cancers, and cancers associated with tobacco use and human papillomavirus (HPV). Rural-urban comparisons were made by demographic, geographic, and socioeconomic characteristics for 2009 to 2013. Trends were analyzed for 1995 to 2013. Combined cancers incidence rates were generally higher in urban populations, except for the South, although the urban decline in incidence rate was greater than in rural populations (10.2% vs. 4.8%, respectively). Rural cancer disparities included higher rates of tobacco-associated, HPV-associated, lung and bronchus, cervical, and colorectal cancers across most population groups. Furthermore, HPV-associated cancer incidence rates increased in rural areas (APC = 0.724, < 0.05), while temporal trends remained stable in urban areas. Cancer rates associated with modifiable risks-tobacco, HPV, and some preventive screening modalities (e.g., colorectal and cervical cancers)-were higher in rural compared with urban populations. Population-based, clinical, and/or policy strategies and interventions that address these modifiable risk factors could help reduce cancer disparities experienced in rural populations.
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http://dx.doi.org/10.1158/1055-9965.EPI-17-0430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787045PMC
November 2018

Single nucleotide variant in Nucleoporin 107 may be predictive of sensitivity to chemotherapy in patients with ovarian cancer.

Pharmacogenet Genomics 2017 07;27(7):264-269

aDepartment of Urology, Henry Ford Health System, Detroit, Michigan bCenter for Clinical Research Departments of cMedical Microbiology, Immunology and Cell Biology dObstetrics and Gynecology eSimmons Cancer Institute, Southern Illinois University School of Medicine, Springfield, Illinois, USA.

Background: Alterations in nuclear pore complex (NPC) genes have been previously associated with response to chemotherapy. Using agnostic exome sequencing, we envisioned that new alleles in NPC genes, predictive of sensitivity to platinum treatment, could be discovered.

Methods: Twenty-two platinum-sensitive and six platinum-resistant ovarian cancer patients were tested. Platinum sensitivity was defined as disease-free survival greater than 6 months. Next-generation sequencing of exomes was used to compare platinum-sensitive and platinum-resistant patients. Single nucleotide variants (SNVs) associated with platinum sensitivity in NPC genes (n=30 genes) were identified.

Results: SNVs in three NPC genes were associated with response to platinum on univariate analysis. SNV rs79419059 (10T>C) in Nucleoporin 107 (Nup107) was associated with platinum resistance (P=0.0061), whereas rs2302811 (3662-4A>G) in Nucleoporin 188 (Nup188) and rs77246077 (3420-67T>A) in Nucleoporin 214 (Nup214) were associated with platinum sensitivity (P=0.0483 and 0.0091, respectively). Controlling for other confounders, multivariate age-adjusted Cox proportional hazard analysis showed rs79419059 to be significantly associated with platinum resistance (odds ratio: 4.519, 95% confidence interval: 1.317-15.501, P=0.0457).

Conclusion: We identified a variant in the 3'-UTR region Nup107 unique to sensitivity to platinum in ovarian cancer. With validation of this variant, it is possible that a new marker predictive of patient response may be identified.
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http://dx.doi.org/10.1097/FPC.0000000000000288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298606PMC
July 2017

Evaluation of the cytotoxicity of the Bithionol - cisplatin combination in a panel of human ovarian cancer cell lines.

BMC Cancer 2017 01 13;17(1):49. Epub 2017 Jan 13.

Division of Gynecologic Oncology; Department of Obstetrics and Gynecology, Southern Illinois University School of Medicine, Springfield, IL, USA.

Background: Combination drug therapy appears a promising approach to overcome drug resistance and reduce drug-related toxicities in ovarian cancer treatments. In this in vitro study, we evaluated the antitumor efficacy of cisplatin in combination with Bithionol (BT) against a panel of ovarian cancer cell lines with special focus on cisplatin-sensitive and cisplatin-resistant cell lines. The primary objectives of this study are to determine the nature of the interactions between BT and cisplatin and to understand the mechanism(s) of action of BT-cisplatin combination.

Methods: The cytotoxic effects of drugs either alone or in combination were evaluated using presto-blue assay. Cellular reactive oxygen species were measured by flow cytometry. Immunoblot analysis was carried out to investigate changes in levels of cleaved PARP, XIAP, bcl-2, bcl-xL, p21 and p27. Luminescent and colorimetric assays were used to test caspases 3/7 and ATX activity.

Results: The efficacy of the BT-cisplatin combination depends upon the cell type and concentrations of cisplatin and BT. In cisplatin-sensitive cell lines, BT and cisplatin were mostly antagonistic except when used at low concentrations, where synergy was observed. In contrast, in cisplatin-resistant cells, BT-cisplatin combination treatment displayed synergistic effects at most of the drug ratios/concentrations. Our results further revealed that the synergistic interaction was linked to increased reactive oxygen species generation and apoptosis. Enhanced apoptosis was correlated with loss of pro-survival factors (XIAP, bcl-2, bcl-xL), expression of pro-apoptotic markers (caspases 3/7, PARP cleavage) and enhanced cell cycle regulators p21 and p27.

Conclusion: In cisplatin-resistant cell lines, BT potentiated cisplatin-induced cytotoxicity at most drug ratios via enhanced ROS generation and modulation of key regulators of apoptosis. Low doses of BT and cisplatin enhanced efficiency of cisplatin treatment in all the ovarian cancer cell lines tested. Our results suggest that novel combinations such as BT and cisplatin might be an attractive therapeutic approach to enhance ovarian cancer chemosensitivity. Combining low doses of cisplatin with subtherapeutic doses of BT can ultimately lead to the development of an innovative combination therapy to reduce/prevent the side effects normally occurring when high doses of cisplatin are administered.
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http://dx.doi.org/10.1186/s12885-016-3034-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5234112PMC
January 2017

Assessment of the antitumor potential of Bithionol in vivo using a xenograft model of ovarian cancer.

Anticancer Drugs 2016 07;27(6):547-59

aDepartment of Obstetrics and Gynecology, Division of Gynecology Oncology bLaboratory Animal Resource Center, Division of Laboratory Animal Medicine, Southern Illinois University School of Medicine, Springfield, Illinois, USA.

In terms of the concept of 'drug repurposing', we focused on pharmaceutical-grade Bithionol (BT) as a therapeutic agent against ovarian cancer. Our recent in-vitro study provides preclinical data suggesting a potential therapeutic role for BT against recurrent ovarian cancer. BT was shown to cause cell death by caspases-mediated apoptosis. The present preliminary study further explores the antitumor potential of pharmaceutical-grade BT in an in-vivo xenograft model of human ovarian cancer. Nude Foxn1 mice bearing SKOV-3 human ovarian tumor xenografts were treated with titrated doses of BT and the therapeutic efficacy of pharmaceutical BT was determined using bioluminescence imaging. BT-induced changes in cell proliferation and apoptosis were evaluated by Ki-67 immunochemical staining and TUNEL assay. The effect of BT on autotaxin levels in serum, ascitic fluid, and tumor tissue was assessed by colorimetric and western blot techniques. BT treatment did not show antitumor potential or enhanced survival time at any of the doses tested. No apparent signs of toxicity were observed with any of the doses tested. Immunohistological analysis of tumor sections did not indicate a significant decrease in cellular proliferation (Ki-67 assay). An increase in apoptosis (by TUNEL assay) was observed in all BT-treated mice compared with vehicle-treated mice. Although BT did not show significant antitumor activity in the present study, the ability of BT to induce apoptosis still makes it a promising therapeutic agent. Further confirmatory and optimization studies are essential to enhance the therapeutic effects of BT.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053334PMC
http://dx.doi.org/10.1097/CAD.0000000000000364DOI Listing
July 2016

Expression profiling of primary and metastatic ovarian tumors reveals differences indicative of aggressive disease.

PLoS One 2014 14;9(4):e94476. Epub 2014 Apr 14.

Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Southern Illinois University School of Medicine, Springfield, Illinois, United States of America.

The behavior and genetics of serous epithelial ovarian cancer (EOC) metastasis, the form of the disease lethal to patients, is poorly understood. The unique properties of metastases are critical to understand to improve treatments of the disease that remains in patients after debulking surgery. We sought to identify the genetic and phenotypic landscape of metastatic progression of EOC to understand how metastases compare to primary tumors. DNA copy number and mRNA expression differences between matched primary human tumors and omental metastases, collected at the same time during debulking surgery before chemotherapy, were measured using microarrays. qPCR and immunohistochemistry validated findings. Pathway analysis of mRNA expression revealed metastatic cancer cells are more proliferative and less apoptotic than primary tumors, perhaps explaining the aggressive nature of these lesions. Most cases had copy number aberrations (CNAs) that differed between primary and metastatic tumors, but we did not detect CNAs that are recurrent across cases. A six gene expression signature distinguishes primary from metastatic tumors and predicts overall survival in independent datasets. The genetic differences between primary and metastatic tumors, yet common expression changes, suggest that the major clone in metastases is not the same as in primary tumors, but the cancer cells adapt to the omentum similarly. Together, these data highlight how ovarian tumors develop into a distinct, more aggressive metastatic state that should be considered for therapy development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0094476PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986100PMC
January 2015

Bithionol inhibits ovarian cancer cell growth in vitro - studies on mechanism(s) of action.

BMC Cancer 2014 Feb 4;14:61. Epub 2014 Feb 4.

Division of Gynecologic Oncology; Department of Obstetrics and Gynecology, Southern Illinois University School of Medicine, Springfield, IL, USA.

Background: Drug resistance is a cause of ovarian cancer recurrence and low overall survival rates. There is a need for more effective treatment approaches because the development of new drug is expensive and time consuming. Alternatively, the concept of 'drug repurposing' is promising. We focused on Bithionol (BT), a clinically approved anti-parasitic drug as an anti-ovarian cancer drug. BT has previously been shown to inhibit solid tumor growth in several preclinical cancer models. A better understanding of the anti-tumor effects and mechanism(s) of action of BT in ovarian cancer cells is essential for further exploring its therapeutic potential against ovarian cancer.

Methods: The cytotoxic effects of BT against a panel of ovarian cancer cell lines were determined by Presto Blue cell viability assay. Markers of apoptosis such as caspases 3/7, cPARP induction, nuclear condensation and mitochondrial transmembrane depolarization were assessed using microscopic, FACS and immunoblotting methods. Mechanism(s) of action of BT such as cell cycle arrest, reactive oxygen species (ROS) generation, autotaxin (ATX) inhibition and effects on MAPK and NF-kB signalling were determined by FACS analysis, immunoblotting and colorimetric methods.

Results: BT caused dose dependent cytotoxicity against all ovarian cancer cell lines tested with IC50 values ranging from 19 μM - 60 μM. Cisplatin-resistant variants of A2780 and IGROV-1 have shown almost similar IC50 values compared to their sensitive counterparts. Apoptotic cell death was shown by expression of caspases 3/7, cPARP, loss of mitochondrial potential, nuclear condensation, and up-regulation of p38 and reduced expression of pAkt, pNF-κB, pIκBα, XIAP, bcl-2 and bcl-xl. BT treatment resulted in cell cycle arrest at G1/M phase and increased ROS generation. Treatment with ascorbic acid resulted in partial restoration of cell viability. In addition, dose and time dependent inhibition of ATX was observed.

Conclusions: BT exhibits cytotoxic effects on various ovarian cancer cell lines regardless of their sensitivities to cisplatin. Cell death appears to be via caspases mediated apoptosis. The mechanisms of action appear to be partly via cell cycle arrest, ROS generation and inhibition of ATX. The present study provides preclinical data suggesting a potential therapeutic role for BT against recurrent ovarian cancer.
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http://dx.doi.org/10.1186/1471-2407-14-61DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3922745PMC
February 2014

PT19c, Another Nonhypercalcemic Vitamin D2 Derivative, Demonstrates Antitumor Efficacy in Epithelial Ovarian and Endometrial Cancer Models.

Genes Cancer 2013 Nov;4(11-12):524-34

Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants' Hospital, Brown University, Providence, RI, USA.

Hypercalcemia remains a major impediment to the clinical use of vitamin D in cancer treatment. Approaches to remove hypercalcemia and development of nonhypercalcemic agents can lead to the development of vitamin D-based therapies for treatment of various cancers. In this report, in vitro and in vivo anticancer efficacy, safety, and details of vitamin D receptor (VDR) interactions of PT19c, a novel nonhypercalcemic vitamin D derived anticancer agent, are described. PT19c was synthesized by bromoacetylation of PTAD-ergocalciferol adduct. Broader growth inhibitory potential of PT19c was evaluated in a panel of chemoresistant breast, renal, ovarian, lung, colon, leukemia, prostate, melanoma, and central nervous system cancers cell line types of NCI60 cell line panel. Interactions of PT19c with VDR were determined by a VDR transactivation assay in a VDR overexpressing VDR-UAS-bla-HEK293 cells, in vitro VDR-coregulator binding, and molecular docking with VDR-ligand binding domain (VDR-LBD) in comparison with calcitriol. Acute toxicity of PT19c was determined in nontumored mice. In vivo antitumor efficacy of PT19c was determined via ovarian and endometrial cancer xenograft experiments. Effect of PT19c on actin filament organization and focal adhesion formation was examined by microscopy. PT19c treatment inhibited growth of chemoresistant NCI60 cell lines (log10GI50 ~ -4.05 to -6.73). PT19c (10 mg/kg, 35 days) reduced growth of ovarian and endometrial xenograft tumor without hypercalcemia. PT19c exerted no acute toxicity up to 400 mg/kg (QDx1) in animals. PT19c showed weak VDR antagonism, lack of VDR binding, and inverted spatial accommodation in VDR-LBD. PT19c caused actin filament dysfunction and inhibited focal adhesion in SKOV-3 cells. PT19c is a VDR independent nonhypercalcemic vitamin D-derived agent that showed noteworthy safety and efficacy in ovarian and endometrial cancer animal models and inhibited actin organization and focal adhesion in ovarian cancer cells.
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http://dx.doi.org/10.1177/1947601913507575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3877664PMC
November 2013

Identification of ovarian cancer metastatic miRNAs.

PLoS One 2013 12;8(3):e58226. Epub 2013 Mar 12.

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island, USA.

Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2-4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, up-regulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058226PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3595263PMC
September 2013

7 Methyl indole ethyl isothiocyanate causes ROS mediated apoptosis and cell cycle arrest in endometrial cancer cells.

Gynecol Oncol 2012 Aug 2;126(2):252-8. Epub 2012 May 2.

Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants Hospital of Rhode Island, Alpert Medical School, Brown University, Providence, RI 02905, USA.

Objective: Chemotherapy options for advanced endometrial cancer are limited and newer therapeutic agents are urgently needed. This study describes the therapeutic potential of 7 Methyl-indole ethyl isothiocyanate (7Me-IEITC) in endometrial cancer cell lines.

Methods: 7Me-IEITC was synthesized in our laboratory. The cell viability of 7Me-IEITC treated ECC-1 and KLE endometrial cancer cell was determined by MTS assay. Morphology and apoptosis were further confirmed by DAPI-staining and TUNEL assay. The measurement of reactive oxygen species (ROS), mitochondrial transmembrane depolarization potential (ΔΨm) and cell cycle phase was determined by FACS analysis. Expression of proteins involved in apoptosis, survival and cell-cycle progression was analyzed by Western blotting.

Results: 7Me-IEITC reduced the viability of the ECC-1 and KLE cancer cell-lines (IC(50)~2.5-10 μM) in a dose dependent fashion. 7Me-IEITC treatment caused mitochondrial transmembrane potential reduction, elevated the production of ROS, leading to activation of apoptosis in endometrial cancer KLE and ECC-1 cells. 7Me-IEITC treatment activated Bad, suppressed Bcl2 phosphorylation followed by PARP-1 deactivation and caspase 3 and 7 activation. 7Me-IEITC treatment arrested the progression of KLE cells in S-phase and caused CDC25 and cyclin-D1 downregulation. Pre-treatment with ascorbic acid abrogated 7Me-IEITC induced apoptosis in ECC-1 and KLE cells, suggesting that 7Me-IEITC mediated cytotoxicity is primarily through ROS production.

Conclusion: 7Me-IEITC demonstrated promising cytotoxic effects in endometrial cancer cell line model.
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http://dx.doi.org/10.1016/j.ygyno.2012.04.041DOI Listing
August 2012

Efficacy of a non-hypercalcemic vitamin-D2 derived anti-cancer agent (MT19c) and inhibition of fatty acid synthesis in an ovarian cancer xenograft model.

PLoS One 2012 3;7(4):e34443. Epub 2012 Apr 3.

Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants' Hospital of Rhode Island, Alpert Medical School, Brown University, Providence, Rhode Island, United States of America.

Background: Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models.

Methodology/principal Finding: Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c-VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein.

Significance: Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034443PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3317945PMC
August 2012

Tetrathiomolybdate sensitizes ovarian cancer cells to anticancer drugs doxorubicin, fenretinide, 5-fluorouracil and mitomycin C.

BMC Cancer 2012 Apr 13;12:147. Epub 2012 Apr 13.

Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants Hospital, Alpert Medical School, Brown University, Providence, RI 02905, USA.

Background: Our recent study showed that tetrathiomolybdate (TM), a drug to treat copper overload disorders, can sensitize drug-resistant endometrial cancer cells to reactive oxygen species (ROS)-generating anticancer drug doxorubicin. To expand these findings in the present study we explore TM efficacy in combination with a spectrum of ROS-generating anticancer drugs including mitomycin C, fenretinide, 5-fluorouracil and doxorubicin in ovarian cancer cells as a model system.

Methods: The effects of TM alone or in combination with doxorubicin, mitomycin C, fenretinide, or 5-fluorouracil were evaluated using a sulforhodamine B assay. Flow cytometry was used to detect the induction of apoptosis and ROS generation. Immunoblot analysis was carried out to investigate changes in signaling pathways.

Results: TM potentiated doxorubicin-induced cytotoxicity and modulated key regulators of apoptosis (PARP, caspases, JNK and p38 MAPK) in SKOV-3 and A2780 ovarian cancer cell lines. These effects were linked to the increased production of ROS, as shown in SKOV-3 cells. ROS scavenging by ascorbic acid blocked the sensitization of cells by TM. TM also sensitized SKOV-3 to mitomycin C, fenretinide, and 5-fluorouracil. The increased cytotoxicity of these drugs in combination with TM was correlated with the activity of ROS, loss of a pro-survival factor (e.g. XIAP) and the appearance of a pro-apoptotic marker (e.g. PARP cleavage).

Conclusions: Our data show that TM increases the efficacy of various anticancer drugs in ovarian cancer cells in a ROS-dependent manner.
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http://dx.doi.org/10.1186/1471-2407-12-147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3353246PMC
April 2012

WNT7A regulates tumor growth and progression in ovarian cancer through the WNT/β-catenin pathway.

Mol Cancer Res 2012 Mar 9;10(3):469-82. Epub 2012 Jan 9.

Department of Physiology, Southern Illinois University School of Medicine, 1135 Lincoln Drive, Carbondale, IL 62901, USA.

Abnormal activation the WNT/β-catenin signaling pathway has been associated with ovarian carcinomas, but a specific WNT ligand and pertinent downstream mechanisms are not fully understood. In this study, we found abundant WNT7A in the epithelium of serous ovarian carcinomas, but not detected in borderline and benign tumors, normal ovary, or endometrioid carcinomas. To characterize the role of WNT7A in ovarian tumor growth and progression, nude mice were injected either intraperitoneally or subcutaneously with WNT7A knocked down SKOV3.ip1 and overexpressed SKOV3 cells. In the intraperitoneal group, mice receiving SKOV3.ip1 cells with reduced WNT7A expression developed significantly fewer tumor lesions. Gross and histologic examination revealed greatly reduced invasion of WNT7A knockdown cells into intestinal mesentery and serosa compared with the control cells. Tumor growth was regulated by loss or overexpression of WNT7A in mice receiving subcutaneous injection as well. In vitro analysis of cell function revealed that cell proliferation, adhesion, and invasion were regulated by WNT7A. The activity of the T-cell factor/lymphoid enhancer factor (TCF/LEF) reporter was stimulated by overexpression of WNT7A in ovarian cancer cells. Cotransfection with WNT7A and FZD5 receptor further increased activity, and this effect was inhibited by cotransfection with SFRP2 or dominant negative TCF4. Overexpression of WNT7A stimulated matrix metalloproteinase 7 (MMP7) promoter, and mutation of TCF-binding sites in MMP7 promoter confirmed that activation of MMP7 promoter by WNT7A was mediated by β-catenin/TCF signaling. Collectively, these results suggest that reexpression of WNT7A during malignant transformation of ovarian epithelial cells plays a critical role in ovarian cancer progression mediated by WNT/β-catenin signaling pathway.
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http://dx.doi.org/10.1158/1541-7786.MCR-11-0177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3307825PMC
March 2012

Purified cranberry proanthocyanidines (PAC-1A) cause pro-apoptotic signaling, ROS generation, cyclophosphamide retention and cytotoxicity in high-risk neuroblastoma cells.

Int J Oncol 2012 Jan 6;40(1):99-108. Epub 2011 Oct 6.

Department of Plant Biology, Rutgers University, New Brunswick, NJ 08901, USA.

Optimized purification of oligomeric proanthocyanidines (PAC) from cranberry generated PAC-1A which selectively affected the viability of various neuroblastoma (NB) cell lines representing a spectrum of high-risk NB features. PAC-1A caused a loss of mitochondrial transmembrane depolarization potential (∆Ψm) and increased generation of reactive oxygen species (ROS) which was directly correlated to the modulation of apoptotic marker proteins in SMS-KCNR cells. PAC-1A reduced the expression of pro-survival (Bcl-2, MCL-1, Bcl-xL) and increased levels of pro-apoptotic (Bax, Bad, Bid) Bcl family proteins, upregulated the activity of SAPK/JNK MAPK and downregulated expression or activity of PI3K/AKT/mTOR pathway components. PAC-1A increased the cellular uptake/retention of cyclophosphamide (CP). PAC-1A and CP synergistically increased cytotoxicity and expression of pro-apoptotic markers, reduced cellular glutathione (GSH) and superoxide dismutase (SOD) levels. Additional features of PAC-1A as an anticancer drug as shown in SMS-KCNR NB cells include delay of cell cycle progression and induction of cell death via TNF-family death receptor activity, thus, targeting both the extrinsic and intrinsic pathway of apoptosis. PAC-1A partially blocked the cell cycle in G2/M phase which correlated with a decrease of the G0/G1 subpopulation, upregulation of cyclin D1 and downregulation of CDK6 and p27 expression. In summary, PAC-1A has demonstrated chemotherapeutic potential to treat a broad spectrum of NBs including highly malignant tumors that show resistance to standard chemotherapeutics and apoptotic stimuli.
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http://dx.doi.org/10.3892/ijo.2011.1225DOI Listing
January 2012

Cytotoxic properties of Adamantyl isothiocyanate and potential in vivo metabolite adamantyl-N-acetylcystein in gynecological cancer cells.

Chem Biol Drug Des 2012 Jan 4;79(1):92-103. Epub 2011 Nov 4.

Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants' Hospital, Alpert Medical School, Brown University, Providence, RI 02905, USA.

This study determined the in vitro potential of novel compounds adamantyl-N-acetylcystein and adamantyl isothiocyanate to treat gynecological cancers. Adamantyl-N-acetylcystein is postulated to be an in vivo metabolite of adamantyl isothiocyanate as dietary isothiocyanates are converted to N-acetylcysteine-conjugates. A viability assay suggested that adamantyl isothiocyanate and adamantyl-N-acetylcystein are cytotoxic to cancer cells including gynecological cell lines. A NCI60 cancer cell assay revealed that growth-inhibition and cytotoxicity of adamantyl-N-acetylcystein were cell line, but not tissue type-specific. Cell cycle studies revealed that adamantyl-N-acetylcystein and adamantyl isothiocyanate arrest SKOV-3 ovarian cancer cells in G2/M phase. By TUNEL, immunoblotting, and viability studies employing caspase and p38 mitogen-activated protein kinase inhibitors, we proved that reduction in SKOV-3 viability is a consequence of DNA fragmentation and apoptosis. Cytotoxic action of adamantyl-N-acetylcystein in SKOV-3 and endometrial cancer (ECC-1, RL95-2, AN3CA, and KLE) cells required excess generation of reactive oxygen species which could be blocked by antioxidant co-treatment. Adamantyl-N-acetylcystein treatment led to modified expression or activation of apoptotic and oncogenic proteins such as JNK/SAPK, AKT, XIAP, and EGF-R for SKOV-3 and JNK/SAPK and ERK1/2 for ECC-1 cells. We suggest the further development of adamantyl-N-acetylcystein by sensitizing cells to the drug using signaling inhibitors or redox-modulating agents and by evaluating the drug efficacy in ovarian and endometrial in-vivo tumor models.
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http://dx.doi.org/10.1111/j.1747-0285.2011.01251.xDOI Listing
January 2012

Anti-angiogenic activity of cranberry proanthocyanidins and cytotoxic properties in ovarian cancer cells.

Int J Oncol 2012 Jan 12;40(1):227-35. Epub 2011 Sep 12.

Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants Hospital of Rhode Island, Providence, RI 02905, USA.

Cranberry extracts may provide beneficial health effects in the treatment of various diseases, including cancer. However, the underlying molecular mechanisms of antineoplastic properties are not understood. We report the effect of a proanthocyanidin (PAC)-rich isolate from cranberry (PAC-1) as a therapeutic agent with dual activity to target both ovarian cancer viability and angiogenesis in vitro. PAC-1 treatment of chemotherapy-resistant SKOV-3 cells blocked cell cycle progression through the G2/M phase, increased the generation of reactive oxygen species (ROS), and induced apoptosis through activation of intrinsic and extrinsic pathway components. Cytotoxicity of PAC-1 was partially based on ROS generation and could be blocked by co-treatment with antioxidant glutathione. PAC-1 reduced the cell viability of both SKOV-3 ovarian cancer cells and HUVEC endothelial cells in a dose-dependent manner and blocked the activation of the pro-survival factor AKT. Furthermore, PAC-1 blocked vascular endothelial growth factor (VEGF)-stimulated receptor phosphorylation in endothelial cells, which correlated with the inhibition of endothelial tube formation in vitro. Our findings suggest that PAC-1 exerts potent anticancer and anti-angiogenic properties and that highly purified PAC from cranberry can be further developed to treat ovarian cancer in combinational or single-agent therapy.
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http://dx.doi.org/10.3892/ijo.2011.1198DOI Listing
January 2012

T0901317 inhibits cisplatin-induced apoptosis in ovarian cancer cells [corrected].

Int J Gynecol Cancer 2011 Nov;21(8):1350-6

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI, USA.

Objective: To determine the function of T0901317 in combination treatment with cisplatin in ovarian cancer cells.

Methods: We screened the effects of 3 nuclear hormone receptor ligands on cell viability in a panel of ovarian cancer cell lines. T0901317 regulation of apoptosis and cell cycle regulators was determined when applied as a single agent or in combination with cisplatin.

Results: Surprisingly, the liver X receptor agonist T0901317 had no significant effects on a panel of 7 ovarian cancer cell lines as a single agent. T0901317 does, however, significantly decrease cisplatin efficacy in at least 3 ovarian cancer cell lines. T0901317 reduces cisplatin-induced apoptosis and reverses cisplatin-induced expression of cell cycle regulators. T0901317 seems to work in a liver X receptor-, pregnane X receptor-, and farnesoid X receptor-independent manner, as agonists of these nuclear hormone receptors did not show similar effects. Interestingly, in the A2780-cp drug-resistant cell line, the effect of T0901317 is lost, suggesting that the pathways stimulated by T0901317 to reduce cisplatin efficacy could be inherently active features of the selected resistance.

Conclusions: Together, these data suggest that T0901317 inhibits cisplatin in some ovarian cancer cells. These data provide an avenue to investigate when T0901317 may be acting to promote tumor survival and drug resistance through control of apoptosis and when it may be acting as an antitumor agent as has been previously reported.
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http://dx.doi.org/10.1097/IGC.0b013e318228f558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203312PMC
November 2011

Evaluation of the first Ergocalciferol-derived, non hypercalcemic anti-cancer agent MT19c in ovarian cancer SKOV-3 cell lines.

Gynecol Oncol 2011 Nov 30;123(2):370-8. Epub 2011 Jul 30.

Department of Obstetrics and Gynecology, Alpert Medical School, Brown University, Providence, RI 02905, USA.

Objective: In human trials calcitriol and its analogs displayed unacceptable systemic toxicities including hypercalcemia. This study was designed to evaluate a novel non-hypercalcemic vitamin-D derivative (MT19c) and its anticancer effects in cultured ovarian cancer cell model.

Methods: We modified the Ergocalciferol structure to generate MT19c, a heterocyclic vitamin-D derivative. Hypercalcemic liabilities of MT19c were assessed by estimating the blood calcium levels in drug treated animals. VDR agonistic or antagonistic properties of MT19c were determined via a VDR-coactivator binding assay. The anticancer effects of MT19c were evaluated by (i) cytotoxicity studies in cancer cell lines and the National Cancer Institute (NCI(60)) cell lines, (ii) identification of apoptosis markers by microscopy and western blots, (iii) cell cycle analysis, and (iv) by studying the insulin receptor substrate-1/2 (IRS1/2) signaling in ovarian cancer cells (SKOV-3) by western blotting.

Results: MT19c treatment did not cause hypercalcemia in mice and showed minor VDR antagonistic activity. In a NCI(60) screen MT19c revealed cell-type specific growth inhibition. MT19c displayed superior cytotoxicity to cisplatin, calcitriol, EB1089 and Iressa in SKOV-3 cell-lines and was comparable to Taxol in our in vitro assays. In SKOV-3 cells MT19c showed caspase dependent apoptosis, DNA fragmentation and cell cycle arrest. MT19c did not alter VDR but downregulated the IGFR/IRS-1/2-MEK-ras-ERK1/2-pathway via activated TNFα-receptor/SAPK/JNK component.

Conclusion: Our results demonstrate how structural optimization of the vitamin-D scaffold leads to identification of a non-hypercalcemic compound MT19c which exerts cytotoxicity in vitro based on a VDR-independent signaling pathway and displays potent anti-cancer activity in ovarian cancer cell models.
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http://dx.doi.org/10.1016/j.ygyno.2011.07.002DOI Listing
November 2011

Integrated genomics of ovarian xenograft tumor progression and chemotherapy response.

BMC Cancer 2011 Jul 22;11:308. Epub 2011 Jul 22.

Molecular Therapeutics Laboratory, Program in Women’s Oncology, Department of Obstetrics and Gynecology, Women and Infants7 Hospital, Alpert Medical School of Brown University, Providence, RI 02905, USA.

Background: Ovarian cancer is the most deadly gynecological cancer with a very poor prognosis. Xenograft mouse models have proven to be one very useful tool in testing candidate therapeutic agents and gene function in vivo. In this study we identify genes and gene networks important for the efficacy of a pre-clinical anti-tumor therapeutic, MT19c.

Methods: In order to understand how ovarian xenograft tumors may be growing and responding to anti-tumor therapeutics, we used genome-wide mRNA expression and DNA copy number measurements to identify key genes and pathways that may be critical for SKOV-3 xenograft tumor progression. We compared SKOV-3 xenografts treated with the ergocalciferol derived, MT19c, to untreated tumors collected at multiple time points. Cell viability assays were used to test the function of the PPARγ agonist, Rosiglitazone, on SKOV-3 cell growth.

Results: These data indicate that a number of known survival and growth pathways including Notch signaling and general apoptosis factors are differentially expressed in treated vs. untreated xenografts. As tumors grow, cell cycle and DNA replication genes show increased expression, consistent with faster growth. The steroid nuclear receptor, PPARγ, was significantly up-regulated in MT19c treated xenografts. Surprisingly, stimulation of PPARγ with Rosiglitazone reduced the efficacy of MT19c and cisplatin suggesting that PPARγ is regulating a survival pathway in SKOV-3 cells. To identify which genes may be important for tumor growth and treatment response, we observed that MT19c down-regulates some high copy number genes and stimulates expression of some low copy number genes suggesting that these genes are particularly important for SKOV-3 xenograft growth and survival.

Conclusions: We have characterized the time dependent responses of ovarian xenograft tumors to the vitamin D analog, MT19c. Our results suggest that PPARγ promotes survival for some ovarian tumor cells. We propose that a combination of regulated expression and copy number can identify genes that are likely important for chemotherapy response. Our findings suggest a new approach to identify candidate genes that are critical for anti-tumor therapy.
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http://dx.doi.org/10.1186/1471-2407-11-308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3155912PMC
July 2011

Organometallic iron(III)-salophene exerts cytotoxic properties in neuroblastoma cells via MAPK activation and ROS generation.

PLoS One 2011 Apr 29;6(4):e19049. Epub 2011 Apr 29.

Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants' Hospital, Alpert Medical School, Brown University, Providence, Rhode Island, United States of America.

The objective of the present study was to investigate the specific effects of Iron(III)-salophene (Fe-SP) on viability, morphology, proliferation, cell cycle progression, ROS generation and pro-apoptotic MAPK activation in neuroblastoma (NB) cells. A NCI-DTP cancer screen revealed that Fe-SP displayed high toxicity against cell lines of different tumor origin but not tumor type-specificity. In a viability screen Fe-SP exhibited high cytotoxicity against all three NB cell lines tested. The compound caused cell cycle arrest in G1 phase, suppression of cells progressing through S phase, morphological changes, disruption of the mitochondrial membrane depolarization potential, induction of apoptotic markers as well as p38 and JNK MAPK activation, DNA degradation, and elevated generation of reactive oxygen species (ROS) in SMS-KCNR NB cells. In contrast to Fe-SP, non-complexed salophene or Cu(II)-SP did not raise ROS levels in NB or SKOV-3 ovarian cancer control cells. Cytotoxicity of Fe-SP and activation of caspase-3, -7, PARP, pro-apoptotic p38 and JNK MAPK could be prevented by co-treatment with antioxidants suggesting ROS generation is the primary mechanism of cytotoxic action. We report here that Fe-SP is a potent growth-suppressing and cytotoxic agent for in vitro NB cell lines and, due to its high tolerance in previous animal toxicity studies, a potential therapeutic drug to treat NB tumors in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019049PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084742PMC
April 2011

Oral RKS262 reduces tumor burden in a neuroblastoma xenograft animal model and mediates cytotoxicity through SAPK/JNK and ROS activation in vitro.

Cancer Biol Ther 2011 Jun 15;11(12):1036-45. Epub 2011 Jun 15.

Women and Infants' Hospital, Department of Obstetrics and Gynecology, Alpert Medical School of Brown University, Providence, RI, USA.

Patients diagnosed with high-risk neuroblastoma (NB), an extracranial solid tumor in children, have metastases and low survival (30%) despite aggressive multi-modal therapy. Therefore new therapies are urgently needed. We show significant in vitro and in vivo antitumor efficacy of RKS262 in NB. RKS262 showed superior cytotoxicity (IC(50) = 6-25 μM) against six representative NB cell lines compared to its parent analog Nifurtimox (currently in phase 2). Pre-formulated RKS262 (150 mg/kg/daily) pellets administered orally, suppressed tumor growth (60%, p = 0.021) in NB xenograft mice within 28 days. RKS262-treated SMSKCNR cells showed TUNEL-positive DNA nicks and activation of ROS, MAPKs (SAPK/JNK), caspase-3, and p53, along with suppression of the IGF-1R/PI3K/PKC pathway and the Bcl2 family of proteins. RKS262 caused G(2)/M-phase arrest and suppressed cdc-2, cyclin B1, p21, and cyclin D1/D4 expression. N-acetyl-cysteine (NAC; 10 mM) pre-treatment rescued cell viability of RKS262 (23 µM)-treated SMSKCNR cells, and pre-treatment with ascorbic acid (100 μM) and a MAPK inhibitor SB203580 (20 μM) reversed SAPK/JNK, caspase-3 activation, PARP-1 cleavage, and suppression of IGF-1R, PI3K, and PKC phosphorylation. Further, treatment with exogenous BDNF (50 nM) did not suppress SAPK/JNK or ROS activation due to RKS262. Rather, BDNF (50 nM), EGF (100 nM) and IGF-1 (100 nM) co-treatment with RKS262 induced a remarkable S-phase arrest rather than a G(2)/M phase arrest when RKS262 was used alone. In summary, RKS262 shows oral efficacy in NB xenograft animals, and induces apoptosis in vitro in SMSKCNR cells via cell cycle arrest, MAPK and ROS activation, and suppression of IGF-1R/PI3K/PKC and Bcl2 family proteins in a growth factor (BDNF/EGF/IGF-1)-independent fashion.
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http://dx.doi.org/10.4161/cbt.11.12.15706DOI Listing
June 2011

Tetrathiomolybdate induces doxorubicin sensitivity in resistant tumor cell lines.

Gynecol Oncol 2011 Jul 6;122(1):183-9. Epub 2011 May 6.

Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants Hospital, Alpert Medical School, Brown University, Providence, RI, USA.

Objective: Doxorubicin is a potent anti-cancer agent with efficacy against a broad range of tumors, including endometrial cancer. Doxorubicin produces reactive oxygen species (ROS) resulting in cytotoxicity. Tetrathiomolybdate (TM), a copper-chelating agent, is known to target a cellular antioxidant enzyme copper/zinc-superoxide dismutase. This study tests the hypothesis that TM can modulate antioxidants in tumor cells and render doxorubicin resistant tumor cells sensitive to doxorubicin.

Methods: The anti-cancer activities of doxorubicin and TM, as single agents and in combination, were assessed. Flow cytometric and immunoblot analysis were conducted to investigate the induction of apoptosis and changes in apoptotic signaling pathways.

Results: Doxorubicin-induced growth inhibition was observed in each endometrial cancer cell line (ECC-1, AN3CA, and KLE) tested with cell specificity. ECC-1 and KLE cells were found to have increased resistance to doxorubicin than AN3CA cells. Moreover, doxorubicin mediated apoptosis was greater in the AN3CA cell line than ECC-1 and KLE. The combination of doxorubicin with a sub-cytotoxic level of TM was significantly more effective at inducing apoptosis in doxorubicin resistant cell lines.

Conclusion: Our results highlight the therapeutic potential of TM to sensitize tumor cells to doxorubicin for endometrial cancer treatment.
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http://dx.doi.org/10.1016/j.ygyno.2011.03.035DOI Listing
July 2011

Antitumor activity of nifurtimox is enhanced with tetrathiomolybdate in medulloblastoma.

Int J Oncol 2011 May 10;38(5):1329-41. Epub 2011 Mar 10.

Vermont Cancer Center, University of Vermont College of Medicine, Burlington, VT, USA.

Medulloblastoma, a neuroectodermal tumor arising in the cerebellum, is the most common brain tumor found in children. We recently showed that nifurtimox induces production of reactive oxygen species (ROS) and subsequent apoptosis in neuroblastoma cells both in vitro and in vivo. Tetrathiomolybdate (TM) has been shown to decrease cell proliferation by inhibition of superoxide dismutase-1 (SOD1). Since both nifurtimox and TM increase ROS levels in cells, we investigated whether the combination of nifurtimox and TM would act synergistically in medulloblastoma cell lines (D283, DAOY). Genome-wide transcriptional analysis, by hybridizing RNA isolated from nifurtimox and TM alone or in combination treated and control cells (D283) on Affymetrix exon array gene chips was carried out to further confirm synergy. We show that nifurtimox and TM alone and in combination decreased cell viability and increased ROS levels synergistically. Examination of cell morphology following drug treatment (nifurtimox + TM) and detection of caspase-3 activation via Western blotting indicated that cell death was primarily due to apoptosis. Microarray data from cells treated with nifurtimox and TM validated the induction of oxidative stress, as many Nrf2 target genes (HMOX1, GCLM, SLC7A11 and SRXN1) (p<10(-5)) were upregulated. Other genes related to apoptosis, oxidative stress, DNA damage, protein folding and nucleosome formation were differentially involved in cells following treatment with nifurtimox + TM. Taken together, our results suggest nifurtimox and TM act synergistically in medulloblastoma cells in vitro, and that this combination warrants further studies as a new treatment for medulloblastoma.
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http://dx.doi.org/10.3892/ijo.2011.971DOI Listing
May 2011

A phase 1 study of nifurtimox in patients with relapsed/refractory neuroblastoma.

J Pediatr Hematol Oncol 2011 Jan;33(1):25-30

Department of Pediatrics, Vermont Children's Hospital, Burlington, VT, USA.

The primary aim of this phase 1 study was to determine the maximum tolerated dose (MTD) and evaluate the safety of nifurtimox alone and in combination with cyclophosphamide and topotecan in multiple relapsed/refractory neuroblastoma pediatric patients. The secondary aim was to evaluate the pharmacokinetics of nifurtimox and the treatment response. To these ends, we performed a phase 1 dose escalation trial of daily oral nifurtimox with toxicity monitoring to determine the MTD, followed by 3 cycles of nifurtimox in combination with cyclophosphamide and topotecan. Samples were collected to determine the pharmacokinetic parameters maximum concentration, time at which maximum concentration is reached, and area under the curve between 0 and 8 hours. Treatment response was evaluated by radiographic and radionuclide (I-metaiodobenzylguanidine) imaging, measurement of urinary catecholamines, and clearance of bone marrow disease. We determined the MTD of nifurtimox to be 30 mg/kg/d. The non-dose-limiting toxicities were mainly nausea and neuropathy. The dose-limiting toxicities of 2 patients at 40 mg/kg/d were a grade 3 pulmonary hemorrhage and a grade 3 neuropathy (reversible). Overall, nifurtimox was well tolerated by pediatric patients at a dose of 30 mg/kg/d, and tumor responses were seen both as a single agent and in combination with chemotherapy. A Phase 2 study to determine the antitumor efficacy of nifurtimox is currently underway.
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http://dx.doi.org/10.1097/MPH.0b013e3181f47061DOI Listing
January 2011

Chemotherapeutic effect of calcidiol derivative B3CD in a neuroblastoma xenograft model.

Chem Biol Drug Des 2010 Aug 11;76(2):164-73. Epub 2010 May 11.

Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants' Hospital of RI, Warren Alpert Medical School of Brown University, Providence, RI 02905, USA.

Bromoacetoxy-calcidiol (B3CD), a pro-apoptotic and cytotoxic agent in neuroblastoma (NB) cell lines, displayed therapeutic potential in vivo as an anticancer drug in a NB xenograft mouse model. Tumors of all animals treated intraperitoneally with B3CD went into regression within 10-30 days of treatment, while tumors in control animals grew aggressively. The response mechanisms of NB cells to B3CD in vitro were studied and included differential targeting of cell cycle key regulators p21 and cyclin D1 on the transcriptional and expression level leading to arrest in G0/G1 phase. In contrast to the effect in ovarian cancer cells, B3CD-induced cell death in SMS-KCNR NB cells was only marginally mediated by the p38 MAPK signaling pathway. Signaling induced by exogenous recombinant EGF leads to a partial restoration of the negative effects of B3CD on SMS-KCNR cell proliferation and survival. Upon combinational treatment of SMS-KCNR cells with B3CD and recombinant EGF, the EGF receptor (EGF-R) was highly activated. We suggest future studies to include analysis of the effects of B3CD in combination therapy with pharmacological inhibitors of cell cycle regulators or with EGF-R-targeting inhibitors, -toxins or -antibodies in vitro and their translation into in vivo models of tumor development.
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http://dx.doi.org/10.1111/j.1747-0285.2010.00988.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3024052PMC
August 2010

Lipophilic aroylhydrazone chelator HNTMB and its multiple effects on ovarian cancer cells.

BMC Cancer 2010 Feb 25;10:72. Epub 2010 Feb 25.

Molecular Therapeutics Laboratory, Program in Women's Oncology, Department of Obstetrics and Gynecology, Women and Infants' Hospital of RI, Alpert Medical School of Brown University, Providence, RI 02905, USA.

Background: Metal chelators have gained much attention as potential anti-cancer agents. However, the effects of chelators are often linked solely to their capacity to bind iron while the potential complexation of other trace metals has not been fully investigated. In present study, we evaluated the effects of various lipophilic aroylhydrazone chelators (AHC), including novel compound HNTMB, on various ovarian cancer cell lines (SKOV-3, OVCAR-3, NUTU-19).

Methods: Cell viability was analyzed via MTS cytotoxicity assays and NCI60 cancer cell growth screens. Apoptotic events were monitored via Western Blot analysis, fluorescence microscopy and TUNEL assay. FACS analysis was carried out to study Cell Cycle regulation and detection of intracellular Reactive Oxygen Species (ROS) RESULTS: HNTMB displayed high cytotoxicity (IC50 200-400 nM) compared to previously developed AHC (oVtBBH, HNtBBH, StBBH/206, HNTh2H/315, HNI/311; IC50 0.8-6 microM) or cancer drug Deferoxamine, a hexadentate iron-chelator (IC50 12-25 microM). In a NCI60 cancer cell line screen HNTMB exhibited growth inhibitory effects with remarkable differences in specificity depending on the cell line studied (GI50 10 nM-2.4 microM). In SKOV-3 ovarian cancer cells HNTMB treatment led to chromatin fragmentation and activation of the extrinsic and intrinsic pathways of apoptosis with specific down-regulation of Bcl-2. HNTMB caused delayed cell cycle progression of SKOV-3 through G2/M phase arrest. HNTMB can chelate iron and copper of different oxidation states. Complexation with copper lead to high cytotoxicity via generation of reactive oxygen species (ROS) while treatment with iron complexes of the drug caused neither cytotoxicity nor increased ROS levels.

Conclusions: The present report suggests that both, non-complexed HNTMB as a chelator of intracellular trace-metals as well as a cytotoxic HNTMB/copper complex may be developed as potential therapeutic drugs in the treatment of ovarian and other solid tumors.
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http://dx.doi.org/10.1186/1471-2407-10-72DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836302PMC
February 2010