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1.  Anticancer properties of novel aminoacetonitrile derivative monepantel (ADD 1566) in pre-clinical models of human ovarian cancer 
Monepantel (MPL) is a new anthelmintic agent approved for the treatment of nematode infections in farm animals. As a nematicide, it acts through a nematode-specific nicotinic receptor subtype which explains its exceptional safety in rodents and mammals. In the present study, we evaluated its potential as an anticancer agent. In vitro treatment of epithelial ovarian cancer cells with MPL resulted in reduced cell viability, inhibition of cell proliferation and suppression of colony formation. Proliferation of human ovarian surface epithelial cells and other non-malignant cells were however minimally affected. MPL-induced inhibition was found to be independent of the acetylcholine nicotinic receptor (nAChR) indicating that, its target in cancer cells is probably different from that in nematodes. Analysis of MPL treated cells by flow cytometry revealed G1 phase cell cycle arrest. Accordingly, MPL treated cells expressed reduced levels of cyclins D1 and A whereas cyclin E2 expression was enhanced. Consistent with a G1 phase arrest, cellular levels of cyclin dependent kinases (CDKs) 2 and 4 were lower, whereas expression of CDK inhibitor p27kip was increased. In cells expressing the wild-type p53, MPL treatment led to increased p53 expression. In line with these results, MPL suppressed cellular thymidine incorporation thus impairing DNA synthesis and inducing cleavage of poly (ADP-ribose) polymerase (PARP-1). Combined these pre-clinical findings reveal for the first time the anticancer potential of monepantel.
PMCID: PMC4163619  PMID: 25232496
Monepantel; ovarian cancer; cell cycle; cyclins; PARP-1
2.  Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR/p70S6K signalling pathway 
We have recently shown that the novel anthelmintic drug monepantel (MPL) inhibits growth, proliferation and colony formation, arrests the cell cycle and induces cleavage of PARP-1 in ovarian cancer cell lines. Here we report on the mechanism behind the anticancer properties of MPL. The cytotoxic effect of MPL on ovarian cancer cells (OVCAR-3 and A2780) was investigated employing a panel of tests used for the detection of apoptosis and autophagy. Apoptosis and autophagy were defined by caspase activity, DNA-laddering, Annexin-V and acridine orange (AO) staining. Autophagy markers such as LC3B, SQSTM1/p62 and mammalian target of rapamycin (mTOR) pathway related proteins were assessed by western blotting and ELISA techniques. MPL did not activate caspases 3 or 8, nor did it alter the percentage of Annexin V positive stained cells. Failure to cause DNA laddering and the inability of z-VAD-fmk to block the MPL antiproliferative effects led to the ruling out of apoptosis as the mechanism behind MPL-induced cell death. On the other hand, accumulation of acidic vacuoles with distinct chromatin morphology and an increase in punctuate localization of green fluorescent protein-LC3B, and MPL-induced changes in the expression of SQSTM1/p62 were all indicative of MPL-induced autophagy. Consistent with this, we found inhibition of mTOR phosphorylation leading to suppression of the mTOR/p70S6K signalling pathway. Our findings provide the first evidence to show that MPL triggers autophagy through the deactivation of mTOR/p70S6K signalling pathway.
PMCID: PMC4163620  PMID: 25232497
Monepantel; ovarian cancer; apoptosis; PARP; autophagy; mTOR; p70S6K
3.  An MHC Class II Dependent Activation Loop Between Adipose Tissue Macrophages and CD4+ T cells Controls Obesity-Induced Inflammation 
Cell reports  2014;9(2):605-617.
Summary
An adaptive immune response triggered by obesity is characterized by the activation of adipose tissue CD4+ T cells by unclear mechanisms. We have examined if interactions between adipose tissue macrophages (ATMs) and CD4+ T cells contribute to adipose tissue metainflammation. Intravital microscopy identifies dynamic antigen dependent interactions between ATMs and T cells in visceral fat. Mice deficient in major histocompatibility complex class II (MHCII) showed protection from diet-induced obesity. Deletion of MHCII expression in macrophages led to an adipose tissue specific decrease in the effector/memory CD4+ T cells, attenuation of CD11c+ ATM accumulation, and improvement in glucose intolerance by increasing adipose tissue insulin sensitivity. Ablation experiments demonstrated that the maintenance of proliferating conventional T cells is dependent on signals from CD11c+ ATMs in obese mice. These studies demonstrate the importance of MHC Class II restricted signals from ATMs that regulate adipose tissue T cell maturation and metainflammation.
doi:10.1016/j.celrep.2014.09.004
PMCID: PMC4252867  PMID: 25310975
4.  Flow Cytometry Analyses of Adipose Tissue Macrophages 
Methods in enzymology  2014;537:297-314.
Within adipose tissue, multiple leukocyte interactions contribute to metabolic homeostasis in health as well as to the pathogenesis of insulin resistance with obesity. Adipose tissue macrophages (ATMs) are the predominant leukocyte population in fat and contribute to obesity-induced inflammation. Characterization of ATMs and other leukocytes in the stromal vascular fraction from fat has benefited from the use of flow cytometry and flow-assisted cell sorting techniques. These methods permit the immunophenotyping, quantification, and purification of these unique cell populations from multiple adipose tissue depots in rodents and humans. Proper isolation, quantification, and characterization of ATM phenotypes are critical for understanding their role in adipose tissue function and obesity-induced metabolic diseases. Here, we present the flow cytometry protocols for phenotyping ATMs in lean and obese mice employed by our laboratory.
doi:10.1016/B978-0-12-411619-1.00016-1
PMCID: PMC4244227  PMID: 24480353
5.  Streptozotocin is equally diabetogenic whether administered to fed or fasted mice 
Laboratory animals  2013;47(4):257-265.
Streptozotocin (STZ) is a selective pancreatic β cell toxin used to generate experimental hyperglycemia in rodent models. Several laboratory animal protocols suggest that STZ be administered to fasted rodents to minimize competition between STZ and glucose for low affinity GLUT2 transporters on β cells. However, whether the diabetogenic effects of multiple low dose (MLD)-STZ administration are enhanced by fasting has not been addressed. Given that repeated bouts of fasting can cause undue metabolic stress in mice, we compared the efficacy of MLD-STZ injections (50 mg/kg body weight daily for 5 days) to induce experimental hyperglycemia in both NOD/SCID/γchainnull and C57BL/6J mice that were either ad libitum fed (STZ-Fed) or that had been fasted for 6 h (STZ-Fasted) prior to the time of STZ administration. Both STZ-Fed and STZ-Fasted mice had significantly worse glucose tolerance than vehicle-treated control mice 10 days after initiation of the MLD-STZ regimen. In C57BL/6J mice, fasting glucose levels, serum insulin levels, β cell mass, and glucose disposal during intraperitoneal glucose tolerance tests (IPGTTs) were indistinguishable between STZ-Fed and STZ-Fasted mice 20 days after MLD-STZ. The glucose intolerant phenotypes persisted for 20 weeks thereafter, irrespective of whether C57BL/6J mice were fed or fasted at the time of STZ injections. However, STZ-Fasted C57BL/6J mice experienced significant weight loss during the repeated bouts of fasting/re-feeding that were required to complete the MLD-STZ protocol. In summary, induction of experimental hyperglycemia can be achieved using the MLD-STZ protocol without repeated bouts of fasting, which have the potential to cause metabolic stress in laboratory mice.
doi:10.1177/0023677213489548
PMCID: PMC3773005  PMID: 23760565
streptozotocin; fasting; diabetes; animal protocol refinement
6.  Impact of treatment modality and number of lesions on recurrence and survival outcomes after treatment of colorectal cancer liver metastases 
Background
Ablative strategies have been used to treat and facilitate hepatic resection (HR) in patients with otherwise unresectable colorectal liver metastases (CLM). We evaluated the efficacy of HR, concomitant HR and ablation and isolated ablation on recurrence and survival outcomes after treatment of CLM in patients with 1-4 and ≥5 lesions, respectively.
Methods
A retrospective review of a prospectively collected hepatobiliary surgery database was performed on patients who underwent treatment for isolated CLM between 1990 and 2010. Pre-operative and treatment characteristics were compared between patients who underwent HR, concomitant HR and ablation and ablation alone. The impact of treatment modality on survival and recurrence outcomes was determined.
Results
A total of 701 patients met inclusion criteria; 550 patients (78%) had 1-4 lesions and 151 patients (22%) had ≥5 lesions. Overall median survival for the entire cohort was 35 months with 5- and 10-year survival of 33% and 20%, respectively. Overall median and 5-year recurrence-free survival (RFS) was 13 months and 21%, respectively. For patients with 1-4 lesions, median survival was 37 months with 5-year survival of 36%. Stratified by procedure type, 5-year survival was 41% in patients who underwent HR, 35% in patients who underwent concomitant HR and ablation and 13% in patients who underwent ablation alone (P<0.001). For patients with ≥5 lesions, median survival was 28 months with 5-year survival of 23% without difference between treatment groups (P=0.078).
Conclusions
HR appears to be the most effective strategy for patients with 1-4 lesions. When ≥5 lesions are present, ablative strategies are useful in facilitating HR in otherwise unresectable patients.
doi:10.3978/j.issn.2078-6891.2013.055
PMCID: PMC3904025  PMID: 24490042
Colorectal cancer; hepatectomy; liver metastases; resection; survival; ablation
7.  Adipose Tissue Macrophages Function As Antigen-Presenting Cells and Regulate Adipose Tissue CD4+ T Cells in Mice 
Diabetes  2013;62(8):2762-2772.
The proinflammatory activation of leukocytes in adipose tissue contributes to metabolic disease. How crosstalk between immune cells initiates and sustains adipose tissue inflammation remains an unresolved question. We have examined the hypothesis that adipose tissue macrophages (ATMs) interact with and regulate the function of T cells. Dietary obesity was shown to activate the proliferation of effector memory CD4+ T cells in adipose tissue. Our studies further demonstrate that ATMs are functional antigen-presenting cells that promote the proliferation of interferon-γ–producing CD4+ T cells in adipose tissue. ATMs from lean and obese visceral fat process and present major histocompatibility complex (MHC) class II–restricted antigens. ATMs were sufficient to promote proliferation and interferon-γ production from antigen-specific CD4+ T cells in vitro and in vivo. Diet-induced obesity increased the expression of MHC II and T-cell costimulatory molecules on ATMs in visceral fat, which correlated with an induction of T-cell proliferation in that depot. Collectively, these data indicate that ATMs provide a functional link between the innate and adaptive immune systems within visceral fat in mice.
doi:10.2337/db12-1404
PMCID: PMC3717880  PMID: 23493569
8.  Peritonectomy HIPEC—contemporary results, indications 
doi:10.3978/j.issn.1000-9604.2013.07.03
PMCID: PMC3752351  PMID: 23997522
9.  CX3CR1 Deficiency Does Not Influence Trafficking of Adipose Tissue Macrophages in Mice With Diet-Induced Obesity 
Obesity (Silver Spring, Md.)  2012;20(6):1189-1199.
Adipose tissue macrophages (ATMs) accumulate in fat during obesity and resemble foam cells in atherosclerotic lesions, suggesting that common mechanisms underlie both inflammatory conditions. CX3CR1 and its ligand fractalkine/CX3CL1 contribute to macrophage recruitment and inflammation in atherosclerosis, but their role in obesity-induced adipose tissue inflammation is unknown. Therefore, we tested the hypothesis that CX3CR1 regulates ATM trafficking to epididymal fat and contributes to the development of adipose tissue inflammation during diet-induced obesity. Cx3cl1 and Cx3cr1 expression was induced specifically in epididymal fat from mice fed a high-fat diet (HFD). CX3CR1 was detected on multiple myeloid cells within epididymal fat from obese mice. To test the requirement of CX3CR1 for ATM trafficking and obesity-induced inflammation, Cx3cr1+/GFP and Cx3cr1GFP/GFP mice were fed a HFD. Ly-6cLow monocytes were reduced in lean Cx3cr1GFP/GFP mice; however, HFD-induced monocytosis was comparable between strains. Total ATM content, the ratio of type 1 (CD11c+) to type 2 (CD206+) ATMs, expression of inflammatory markers, and T-cell content were similar in epididymal fat from obese Cx3cr1+/GFP and Cx3cr1GFP/GFP mice. Cx3cr1 deficiency did not prevent the development of obesity-induced insulin resistance or hepatic steatosis. In summary, our data indicate that CX3CR1 is not required for the recruitment or retention of ATMs in epididymal adipose tissue of mice with HFD-induced obesity even though CX3CR1 promotes foam cell formation. This highlights an important point of divergence between the mechanisms regulating monocyte trafficking to fat with obesity and those that contribute to foam cell formation in atherogenesis.
doi:10.1038/oby.2012.7
PMCID: PMC4006981  PMID: 22252034
10.  Does the expression of BCL2 have prognostic significance in malignant peritoneal mesothelioma? 
Background Malignant peritoneal mesothelioma (MPM) is a rare neoplasm of the peritoneal membrane that is causally related to asbestos exposure. Survival after treatment is poor. Current therapy involving hyperthermic intraperitoneal chemotherapy has improved survival in selective patients. In the past, several prognostic factors have been identified in MPM patients and this has prompted the development of new therapies and patient management. Since BCL2, an antiapoptotic oncoprotein, is a favourable prognostic factor in breast cancer, we investigated to determine the significance of BCL2 in MPM. Materials and Methods Forty two archival patient tumour sections embedded in paraffin blocks were sectioned and subjected to immunohistochemistry to detect BCL2. The staining intensity and abundance was classified using standard procedures and classified into two groups (0-4 = low & 5-8 = high expression). The distribution of BCL2 groups was examined in the different clinicopathological categories to determine prognosis using Kaplan–Meier survival analysis. Results: Univariate analysis revealed that in almost all clinicopathological categories, high BCL2 expression predisposed patients to a favourable prognosis. Independent of BCL2 expression, univariate analysis also showed that male gender, sarcomatoid histology, high PCI and age at diagnosis ≥ 60 years were associated poor prognosis. Multivariate analysis indicated that for all tumours, males and females, high BCL2 expression was associated with good prognosis. Further, independent of BCL2, age ≥ 60 years is an unfavourable prognostic factor. Conclusion: Expression of BCL2 may serve to distinguish prognosis within the individual clinicopathological categories. BCL2 is also an independent variable in all tumours, males and females, with high expression being associated with good prognosis.
PMCID: PMC3696537  PMID: 23841030
BCL2; prognosis; peritoneal mesothelioma; survival
11.  Effectiveness of early and aggressive administration of fresh frozen plasma to reduce massive blood transfusion during cytoreductive surgery 
Background
Cytoreductive surgery (CRS) combined with perioperative intraperitoneal chemotherapy (PIC) has been consistently associated with high volume blood loss and red blood cell (RBC) transfusion. This study evaluates the effectiveness of the introduction of a novel protocol to reduce blood loss and subsequent intra-operative transfusion in patients with high volume disease.
Methods
One hundred and thirty-one consecutive patients with high volume disease (peritoneal cancer index ≥16) who underwent CRS and PIC were evaluated. Group I consisted of the sixty patients (46%) treated before June 2006. Group II consistent of the seventy-one (54%) patients treated after June 2006 under the new protocol. The clinical and treatment-related data of patients in the two groups were compared.
Results
Group II was associated with reduced intra-operative red blood cell transfusion (P<0.001), reduced cryoprecipitate transfusion (P=0.020), reduced platelet transfusion (P<0.001), reduced fresh frozen plasma transfusion (P=0.024), reduced operation length (P<0.001), reduced crystalloid administration (P<0.001) and reduced colloid administration (P<0.001). Group II was also associated with increased transfusion of FFP in the first half of the surgical intervention relative to the second half [FFP1st:FFP2nd ratio >1 (P<0.001)] and increased transfusion of RBC in the first half of the surgical intervention relative to the second half [RBC1st:RBC2nd ratio ≥1 (P=0.016)].
Conclusion
Early administration of fresh frozen plasma combined with restrictive fluid resuscitation may reduce overall intra-operative transfusion of RBC and other blood components.
doi:10.3978/j.issn.2078-6891.2012.046
PMCID: PMC3562619  PMID: 23449950
Peritonectomy; cytoreductive surgery; perioperative intraperitoneal chemotherapy; morbidity; mortality; blood transfusion
12.  Approach to Rectal Cancer Surgery 
Rectal cancer is a distinct subset of colorectal cancer where specialized disease-specific management of the primary tumor is required. There have been significant developments in rectal cancer surgery at all stages of disease in particular the introduction of local excision strategies for preinvasive and early cancers, standardized total mesorectal excision for resectable cancers incorporating preoperative short- or long-course chemoradiation to the multimodality sequencing of treatment. Laparoscopic surgery is also increasingly being adopted as the standard rectal cancer surgery approach following expertise of colorectal surgeons in minimally invasive surgery gained from laparoscopic colon resections. In locally advanced and metastatic disease, combining chemoradiation with radical surgery may achieve total eradication of disease and disease control in the pelvis. Evidence for resection of metastases to the liver and lung have been extensively reported in the literature. The role of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for peritoneal metastases is showing promise in achieving locoregional control of peritoneal dissemination. This paper summarizes the recent developments in approaches to rectal cancer surgery at all these time points of the disease natural history.
doi:10.1155/2012/247107
PMCID: PMC3389703  PMID: 22792451
13.  Correction: Neuropeptide Y Is Produced by Adipose Tissue Macrophages and Regulates Obesity-Induced Inflammation 
PLoS ONE  2013;8(9):10.1371/annotation/c2432ace-1dd7-4b02-8980-e4e6c085beba.
doi:10.1371/annotation/c2432ace-1dd7-4b02-8980-e4e6c085beba
PMCID: PMC3812369
14.  Sex-specific selection for MHC variability in Alpine chamois 
Background
In mammals, males typically have shorter lives than females. This difference is thought to be due to behavioural traits which enhance competitive abilities, and hence male reproductive success, but impair survival. Furthermore, in many species males usually show higher parasite burden than females. Consequently, the intensity of selection for genetic factors which reduce susceptibility to pathogens may differ between sexes. High variability at the major histocompatibility complex (MHC) genes is believed to be advantageous for detecting and combating the range of infectious agents present in the environment. Increased heterozygosity at these immune genes is expected to be important for individual longevity. However, whether males in natural populations benefit more from MHC heterozygosity than females has rarely been investigated. We investigated this question in a long-term study of free-living Alpine chamois (Rupicapra rupicapra), a polygynous mountain ungulate.
Results
Here we show that male chamois survive significantly (P = 0.022) longer if heterozygous at the MHC class II DRB locus, whereas females do not. Improved survival of males was not a result of heterozygote advantage per se, as background heterozygosity (estimated across twelve microsatellite loci) did not change significantly with age. Furthermore, reproductively active males depleted their body fat reserves earlier than females leading to significantly impaired survival rates in this sex (P < 0.008). This sex-difference was even more pronounced in areas affected by scabies, a severe parasitosis, as reproductively active males were less likely to survive than females. However, we did not find evidence for a survival advantage associated with specific MHC alleles in areas affected by scabies.
Conclusions
Increased MHC class II DRB heterozygosity with age in males, suggests that MHC heterozygous males survive longer than homozygotes. Reproductively active males appear to be less likely to survive than females most likely because of the energetic challenge of the winter rut, accompanied by earlier depletion of their body fat stores, and a generally higher parasite burden. This scenario renders the MHC-mediated immune response more important for males than for females, which implies a relatively stronger selection pressure on MHC genes in males than in females.
doi:10.1186/1471-2148-12-20
PMCID: PMC3340304  PMID: 22335968
MHC; Sex-specific selection; Heterozygosity advantage; Alpine chamois
15.  Hepatocytes isolated from neoplastic liver-immunomagnetic purging as a new source for transplantation 
AIM: To investigate whether hepatocytes isolated from macroscopically normal liver during hepatic resection for neoplasia could provide a novel source of healthy hepatocytes, including the development of reliable protocols for malignant cells removal from the hepatocyte preparation.
METHODS: Hepatocytes were procured from resected liver of 18 patients with liver tumors using optimised digestion and cell-enrichment protocols. Suspensions of various known quantities of the HT-29 tumor cell line and patient hepatocytes were treated or not with Ep-CAM-antibody-coated immunomagnetic beads in order to investigate the efficacy of tumor-purging by immunomagnetic depletion, using a semi-quantitative RT-PCR method developed to detect tumor cells. Immunomagnetic bead-treated or bead-untreated tumor cell-hepatocyte suspensions were transplanted intra-peritoneally in Balb/C nude mice to assess the rates of tumor development.
RESULTS: Mean viable hepatocyte yield was 9.3 x 106 cells per gram of digested liver with mean viability of 70.5%. Immunomagnetic depletion removed tumor cells to below the RT-PCR detection-threshold of 1 tumor cell in 106 hepatocytes, representing a maximum tumor purging efficacy of greater than 400 000-fold. Transplanted, immunomagnetic bead-purged tumor cell-hepatocyte suspensions did not form peritoneal tumors in Balb/C nude mice. Co-transplantation of hepatocytes with tumor cells did not increase tumorigenesis of the tumor cells.
CONCLUSION: Immunomagnetic depletion appears to be an effective method of purging contaminating tumor cells to below threshold for likely tumorigenesis. Along with improved techniques for isolation of large numbers of viable hepatocytes, normal liver resected for neoplasia has potential as another clinically useful source of hepatocytes for transplantation.
doi:10.3748/wjg.14.5025
PMCID: PMC2742930  PMID: 18763285
Hepatocyte transplantation; Immunomagnetic purging; Isolation of hepatocytes
16.  Ki67-BCL2 index in prognosis of malignant peritoneal mesothelioma 
Background: malignant peritoneal mesothelioma (MPM) is a rare peritoneal mesothelial neoplasm. Ki67 and BCL2 are established prognostic markers in several cancers. High Ki67 expression indicates tumour progression, whilst similar expression of BCL2 retards tumour replication. Traditionally, prognosis in MPM is gauged with a single biomarker assessed separately in a dichotomous manner. Here, we examine prognosis with dual biomarkers incorporated in a model to predict survival. Materials and methods: Forty two MPM archival patient tumours were screened for Ki67 and BCL2 by immunohistochemistry and evaluated using standard methods. Ki67 and BCL2 expression was incorporated into a prognostic model to develop Ki67-BCL2 index. Using this index, three hazard groups were identified (high, medium and low risk). Kaplan-Meier survival analysis was performed to assess the significance of these hazard groups in the various clinicopathological categories. Results: In all clinicopathological categories, high risk group showed poor prognosis compared to low risk group (p = < 0.001). Compared to medium risk, high risk group carried poor prognosis in all tumours, females, epitheloid tumours, peritoneal cancer index (PCI) < 20, ≥ 20, age at diagnosis (AAD) < 60, and ≥ 60 years. Independent of the Ki67-BCL2 index, male, sarcomatoid, PCI ≥ 20 and AAD ≥ 60 were poor prognostic factors. High risk group was an independent poor prognostic factor in all tumours, males, females and age < 60 years. The distribution of high risk: low risk group in male and female was 3: 2 and 2: 3, respectively, indicating a gender difference. Comparing hazard ratios generated by Ki67-BCL2 index to that of either Ki67 or BCL2, as a single prognostic biomarker, there was a reduction of HR values. Conclusion: Ki67-BCL2 index seems to suggest a more sensitive method of predicting prognosis. However, the current model needs further evaluation in an independent large cohort sample.
PMCID: PMC3744020  PMID: 23977450
Ki67; BCl2; prognosis; malignant peritoneal mesothelima
17.  Carbohydrate antigen 19-9 (CA 19-9) is an independent prognostic indicator in pseudomyxoma peritonei post cytoreductive surgery and perioperative intraperitoneal chemotherapy 
Introduction
Pseudomyxoma peritonei (PMP) is characteristically divided into two histopathological subtypes; disseminated peritoneal adenomucinosis (DPAM) and peritoneal mucinous carcinomatosis (PMCA). The latter is associated with a worse prognosis. However, even within the DPAM group, there is a considerable variation in outcome. In this study we investigate the role of baseline serum tumor markers CA 19-9, CEA and CA-125 in further stratifying survival.
Methods
Over 16 years, 218 patients with PMP were treated with cytoreductive surgery (CRS) and perioperative intraperitoneal chemotherapy (PIC) at our institution. A CA-125 level of >35 U/L, CA 19-9 of >40 U/mL and CEA of >3 ng/mL were considered positive or elevated outside the laboratory reference range. The impact of clinicopathologic and treatment-related variables on overall survival (OS) was analyzed with the Kaplan Meier method. Survival curves were compared using the log-rank test. Variables deemed significant by univariate analyses were entered into multivariate analysis using the Cox proportional hazards model.
Results
Within the DPAM group, the 5-year survival of patients who were CA 19-9 positive versus those with normal values were 58% and 90% respectively (P<0.001). Other variables found to negatively impact on OS in univariate analyses were completeness of cytoreduction (CC) score 2/3 (P<0.001), peritoneal cancer index (PCI) >25 (P<0.001) and male gender (P=0.017). In the Cox regression model, only CA 19-9 positivity was found to be an independent prognostic factor for OS (P=0.034). In addition to marker positivity, the absolute level of CA 19-9 was also prognostically significant. In patients with CA 19-9>1,000 U/mL, the 5-year survival was 23%, in contrast to 90% in patients with CA 19-9<100 U/mL (P<0.001).
In the PMCA cohort, only CC-score was found to be associated with OS (P<0.001).
Conclusions
Our study provides relevant prognostic information for the DPAM subtype in staging and prioritizing surgery; as even in apparently indolent disease, some patients have poorer survival. CA 19-9 elevation may also be useful in identifying patients who would potentially benefit from adjuvant therapy and/or closer post-operative surveillance.
The potential role of CA 19-9 in mediating tumor cell adhesion and disease progression in PMP should be further investigated to deepen our understanding of the disease’s inherent biological behavior. If a true relationship exists, CA 19-9 may be a conceivable target for immunotherapy.
doi:10.3978/j.issn.2078-6891.2012.062
PMCID: PMC3635178  PMID: 23730513
Carbohydrate antigen 19-9 (CA 19-9); pseudomyxoma peritonei (PMP); disseminated peritoneal adenomucinosis (DPAM); peritoneal mucinous carcinomatosis (PMCA)
18.  Minocycline Suppresses Interleukine-6, Its Receptor System and Signaling Pathways and Impairs Migration, Invasion and Adhesion Capacity of Ovarian Cancer Cells: In Vitro and In Vivo Studies 
PLoS ONE  2013;8(4):e60817.
Interleukin (IL)-6 has been shown to be a major contributing factor in growth and progression of ovarian cancer. The cytokine exerts pro-tumorigenic activity through activation of several signaling pathways in particular signal transducer and activator of transcription (STAT3) and extracellular signal-regulated kinase (ERK)1/2. Hence, targeting IL-6 is becoming increasingly attractive as a treatment option in ovarian cancer. Here, we investigated the effects of minocycline on IL-6 and its signaling pathways in ovarian cancer. In vitro, minocycline was found to significantly suppress both constitutive and IL-1β or 4-hydroxyestradiol (4-OH-E2)-stimulated IL-6 expression in human ovarian cancer cells; OVCAR-3, SKOV-3 and CAOV-3. Moreover, minocycline down-regulated two major components of IL-6 receptor system (IL-6Rα and gp130) and blocked the activation of STAT3 and ERK1/2 pathways leading to suppression of the downstream product MCL-1. In female nude mice bearing intraperitoneal OVCAR-3 tumors, acute administration (4 and 24 h) of minocycline (30 mg/kg) led to suppression of IL-6. Even single dose of minocycline was effective at significantly lowering plasma and tumor IL-6 levels. In line with this, tumoral expression of p-STAT3, p-ERK1/2 and MCL-1 were decreased in minocycline-treated mice. Evaluation of the functional implication of minocycline on metastatic activity revealed the capacity of minocycline to inhibit cellular migration, invasion and adhesion associated with down-regulation of matrix metalloproteinases (MMP)-2 and 9. Thus, the data suggest a potential role for minocycline in suppressing IL-6 expression and activity. These effects may prove to be an important attribute to the upcoming clinical trials of minocycline in ovarian cancer.
doi:10.1371/journal.pone.0060817
PMCID: PMC3620477  PMID: 23593315
19.  Neuropeptide Y Is Produced by Adipose Tissue Macrophages and Regulates Obesity-Induced Inflammation 
PLoS ONE  2013;8(3):e57929.
Neuropeptide Y (NPY) is induced in peripheral tissues such as adipose tissue with obesity. The mechanism and function of NPY induction in fat are unclear. Given the evidence that NPY can modulate inflammation, we examined the hypothesis that NPY regulates the function of adipose tissue macrophages (ATMs) in response to dietary obesity in mice. NPY was induced by dietary obesity in the stromal vascular cells of visceral fat depots from mice. Surprisingly, the induction of Npy was limited to purified ATMs from obese mice. Significant basal production of NPY was observed in cultured bone marrow derived macrophage and dendritic cells (DCs) and was increased with LPS stimulation. In vitro, addition of NPY to myeloid cells had minimal effects on their activation profiles. NPY receptor inhibition promoted DC maturation and the production of IL-6 and TNFα suggesting an anti-inflammatory function for NPY signaling in DCs. Consistent with this, NPY injection into lean mice decreased the quantity of M1-like CD11c+ ATMs and suppressed Ly6chi monocytes. BM chimeras generated from Npy−/− donors demonstrated that hematopoietic NPY contributes to the obesity-induced induction of Npy in fat. In addition, loss of Npy expression from hematopoietic cells led to an increase in CD11c+ ATMs in visceral fat with high fat diet feeding. Overall, our studies suggest that NPY is produced by a range of myeloid cells and that obesity activates the production of NPY in adipose tissue macrophages with autocrine and paracrine effects.
doi:10.1371/journal.pone.0057929
PMCID: PMC3589443  PMID: 23472120
20.  Combination of Albendazole and 2-Methoxyestradiol significantly improves the survival of HCT-116 tumor-bearing nude mice 
BMC Cancer  2013;13:86.
Background
Albendazole (ABZ) is a microtubule-targeting anthelmintic with a remarkable activity against a variety of human cancer cells. In this study, we examined if the antitumor activity of ABZ could be enhanced by its combination with other microtubule-binding agents.
Methods
The interactions between ABZ and microtubule-binding agents, paclitaxel, vinblastine, colchicine, and 2-methoxyestradiol were characterized using median effect analysis method in HCT-116 colorectal cancer cells and DU145 prostate cancer cell line. The mechanism underlying the synergistic interaction related to tubulin polymerization and apoptosis was then investigated. Finally, the effect of the combination therapy on the survival of HCT-116 tumor-bearing nude mice was evaluated.
Results
Among the tested drugs, a synergistic anti-proliferative effect was observed with the combination of low concentrations of ABZ plus colchicine and ABZ plus 2-methoxyestradiol (2ME). Exploring the mechanism of the interaction between ABZ and 2ME revealed that the combination therapy synergistically activated the extrinsic pathway of apoptosis. Consistent with in vitro results, the combination of low concentration of ABZ with 2ME prolonged the survival of mice-bearing HCT-116 tumors. High concentration of ABZ in combination with 2ME, however, proved to be less effective than ABZ alone.
Conclusions
The combination of low doses of ABZ and 2ME has shown promising results in our pre-clinical model. Additionally, the finding that the combination of two microtubule-binding agents that share the same binding site can act synergistically may lead to the development of new therapeutic strategies in cancer treatment.
doi:10.1186/1471-2407-13-86
PMCID: PMC3606618  PMID: 23432760
Albendazole; 2-Methoxyestradiol; Combination therapy; Microtubule-targeting agents
21.  Assessment of Complement C4 Gene Copy Number Using the Paralog Ratio Test 
Human mutation  2010;31(7):866-874.
The complement C4 locus is in the class III region of the MHC, and exhibits copy number variation. Complement C4 null alleles have shown association with a number of diseases including systemic lupus erythematosus (SLE). However, most studies to date have used protein immunophenotyping and not direct interrogation of the genome to determine C4 null allele status. Moreover, a lack of accurate C4 gene copy number (GCN) estimation and tight linkage disequilibrium across the disease-associated MHC haplotypes has confounded attempts to establish whether or not these associations are causal. We have therefore developed a high through-put paralog ratio test (PRT) in association with two restriction enzyme digest variant ratio tests (REDVRs) to determine total C4 GCN, C4A GCN, and C4B GCN. In the densely genotyped CEU cohort we show that this method is accurate and reproducible when compared to gold standard Southern blot copy number estimation with a discrepancy rate of 9%. We find a broad range of C4 GCNs in the CEU and the 1958 British Birth Cohort populations under study. In addition, SNP-C4 CNV analyses show only moderate levels of correlation and therefore do not support the use of SNP genotypes as proxies for complement C4 GCN.
doi:10.1002/humu.21259
PMCID: PMC3567757  PMID: 20506482
complement C4; CNV; lupus; paralog ratio test
22.  Intraperitoneal chemotherapy in ovarian cancer: a review of tolerance and efficacy 
Purpose
To review the two main approaches of intraperitoneal (IP) chemotherapy delivery in ovarian cancer: postoperative adjuvant IP chemotherapy after cytoreductive surgery (CRS) and intraoperative hyperthermic intraperitoneal chemotherapy (HIPEC).
Methods
A literature search was conducted to identify studies that employed postoperative adjuvant IP chemotherapy after CRS or combined CRS and intraoperative HIPEC in patients with ovarian cancer. Data of interest included chemotherapy protocol, morbidity and mortality, and survival data.
Results
Three large randomized controlled trials comprising 707 patients with advanced ovarian cancer who received postoperative adjuvant IP chemotherapy were reviewed. Morbidity rate ranged from 56% to 94% in IP chemotherapy, and mortality rate ranged from 1% to 2%. Median disease-free survival ranged from 24 to 28 months, and overall survival ranged from 49 to 66 months. Planned chemotherapy completion rates ranged from 42% to 71%. Twenty-four nonrandomized studies that reported HIPEC comprised 1167 patients with both advanced and recurrent ovarian cancer. In patients with advanced ovarian cancer, mortality ranged from 0% to 5%, minor morbidity ranged from 16% to 90%, and major morbidity ranged from 0% to 40%. Median disease-free survival ranged from 13 to 56 months, and overall survival ranged from 14 to 64 months. Survival at 5 years ranged from 35% to 70%. In patients with recurrent ovarian cancer, the mortality rate ranged from 0% to 10%, minor morbidity ranged from 7% to 90%, and major morbidity ranged from 0% to 49%. Median disease-free survival ranged from 13 to 24 months and overall survival from 23 to 49 months. Survival at 5 years ranged from 12% to 54%.
Conclusion
There is level-one evidence suggesting the benefit of postoperative adjuvant intraperitoneal chemotherapy for patients with advanced ovarian cancer after cytoreductive surgery, albeit catheter-related complications resulted after treatment discontinuation. Studies report the use of HIPEC predominantly in the setting of recurrent disease and have demonstrated encouraging results, which merits further investigation in future clinical trials.
doi:10.2147/CMAR.S31070
PMCID: PMC3514065  PMID: 23226073
intraperitoneal chemotherapy; ovarian carcinoma; hyperthermic; intraoperative; cytoreductive surgery
23.  Quality of Life Improvement with Sublingual Immunotherapy: A Prospective Study of Efficacy 
Journal of Allergy  2012;2012:253879.
Due to its excellent safety profile, ease of administration, and economic considerations, sublingual immunotherapy (SLIT) is becoming a preferred form of allergen specific immunotherapy. The efficacy of SLIT is still debated. The purpose of this act of practice trial is to evaluate quality of life outcomes in patients treated with SLIT. Fifty one patients with allergic rhinoconjunctivitis demonstrated by skin testing completed the Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) at initiation, at four months and at 10–12 months of SLIT. Significant improvement (P < 0.05) on six of seven domain categories of the RQLQ questionnaire was noted. Total RQLQ scores also showed significant improvement. This study supports SLIT as a modality effective in controlling allergic symptoms.
doi:10.1155/2012/253879
PMCID: PMC3303579  PMID: 22500181
24.  HAX1 Augments Cell Proliferation, Migration, Adhesion, and Invasion Induced by Urokinase-Type Plasminogen Activator Receptor 
Journal of Oncology  2012;2012:950749.
The urokinase-type plasminogen activator receptor (uPAR) is a cell surface receptor which has a multifunctional task in the process of tumorigenesis including cell proliferation, adhesion, migration, and invasion. Many of the biological functions of uPAR necessitate interactions with other proteins. We have shown previously that uPAR interacts with HAX1 protein (HS-1-associated protein X-1). In the current study, to gain insight into the possible role of HAX1 overexpression in regulation of uPAR signal transduction pathway, several function assays were used. We found that, upon stimulation of uPAR, HAX1 colocalizes with uPAR suggesting a physiological role for HAX1 in the regulation of uPAR signal transduction. HAX1 overexpression augments cell proliferation and migration in uPAR-stimulated cells. Moreover, HAX1 over-expression augmented uPAR-induced cell adhesion to vitronectin as well as cellular invasion. Our results suggest that HAX1 over-expression may underlay a novel mechanism to regulate uPAR-induced functions in cancer cells.
doi:10.1155/2012/950749
PMCID: PMC3270441  PMID: 22315598
25.  Significance of vascular endothelial growth factor in growth and peritoneal dissemination of ovarian cancer 
Cancer Metastasis Reviews  2011;31(1-2):143-162.
Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis which drives endothelial cell survival, proliferation, and migration while increasing vascular permeability. Playing an important role in the physiology of normal ovaries, VEGF has also been implicated in the pathogenesis of ovarian cancer. Essentially by promoting tumor angiogenesis and enhancing vascular permeability, VEGF contributes to the development of peritoneal carcinomatosis associated with malignant ascites formation, the characteristic feature of advanced ovarian cancer at diagnosis. In both experimental and clinical studies, VEGF levels have been inversely correlated with survival. Moreover, VEGF inhibition has been shown to inhibit tumor growth and ascites production and to suppress tumor invasion and metastasis. These findings have laid the basis for the clinical evaluation of agents targeting VEGF signaling pathway in patients with ovarian cancer. In this review, we will focus on VEGF involvement in the pathophysiology of ovarian cancer and its contribution to the disease progression and dissemination.
doi:10.1007/s10555-011-9337-5
PMCID: PMC3350632  PMID: 22101807
Angiogenesis; Bevacizumab; Malignant ascites; Ovarian cancer; Peritoneal carcinomatosis; Vascular endothelial growth factor

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