Future Aspects for Cannabinoids in Breast Cancer Therapy.

Cannabinoids (CBs) from Cannabis sativa provide relief for tumor-associated symptoms (including nausea, anorexia, and neuropathic pain) in the palliative treatment of cancer patients. Additionally, they may decelerate tumor progression in breast cancer patients. Indeed, the psychoactive delta-9-tetrahydrocannabinol (THC), non-psychoactive cannabidiol (CBD) and other CBs inhibited disease progression in breast cancer models. The effects of CBs on signaling pathways in cancer cells are conferred via G-protein coupled CB-receptors (CB-Rs), CB1-R and CB2-R, but also via other receptors, and in a receptor-independent way. THC is a partial agonist for CB1-R and CB2-R; CBD is an inverse agonist for both. In breast cancer, CB1-R expression is moderate, but CB2-R expression is high, which is related to tumor aggressiveness. CBs block cell cycle progression and cell growth and induce cancer cell apoptosis by inhibiting constitutive active pro-oncogenic signaling pathways, such as the extracellular-signal-regulated kinase pathway. They reduce angiogenesis and tumor metastasis in animal breast cancer models. CBs are not only active against estrogen receptor-positive, but also against estrogen-resistant breast cancer cells. In human epidermal growth factor receptor 2-positive and triple-negative breast cancer cells, blocking protein kinase B- and cyclooxygenase-2 signaling via CB2-R prevents tumor progression and metastasis. Furthermore, selective estrogen receptor modulators (SERMs), including tamoxifen, bind to CB-Rs; this process may contribute to the growth inhibitory effect of SERMs in cancer cells lacking the estrogen receptor. In summary, CBs are already administered to breast cancer patients at advanced stages of the disease, but they might also be effective at earlier stages to decelerate tumor progression.

Pharmacokinetics of flucloxacillin and its metabolites in patients with renal failure: Impact on liver toxicity
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OBJECTIVE: The antimicrobial agent flucloxacillin is a potential cause of drug-induced liver disease, but the underlying mechanisms for toxicity have not been fully elucidated. As in-vitro and in-vivo findings suggest that biotransformation products contribute to hepatotoxicity, the purpose of this study was to characterize formation and accumulation of its metabolites in patients with renal failure. METHODS: Twelve intensive care patients undergoing continuous venovenous hemofiltration received 4.0 g flucloxacillin as single and repeated infusion. Blood and dialysate samples were collected and analyzed for flucloxacillin and its metabolites by HPLC. RESULTS: The overall amounts of the flucloxacillin metabolites 5'-hydroxymethylflucloxacillin (5-OH-FX), 5'-hydroxymethylflucloxacillin-penicilloic acid (5-OH-PA), and flucloxacillin-penicilloic acid (FX-PA) produced varied considerably between patients, and accounted for 3.62 - 35.9% of total flucloxacillin concentration (flucloxacillin + metabolites) in the plasma. Clearance rates and sieving coefficients for 5-OH-FX and FX-PA were comparable to that of the parent drug, although removal of 5-OH-PA was decreased. Using an isolated perfused rat liver model we demonstrated that 5-OH-FX reached concentrations in the bile (240.5 ± 84.2 nmoles/mL) that were sufficient to exert cytotoxic effects, unlike either of the two penicilloic acids. CONCLUSIONS: Based on data from perfused rat livers, high biliary concentrations of 5-OH-FX might also be observed in our patients explaining why LDH, bilirubin, and alkaline phosphatase were elevated in up to 8/12 patients after repeated infusion of flucloxacillin. Liver toxicity of flucloxacillin might therefore be observed in patients with renal impairment after continuously elevated 5-OH-FX levels.
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AID/APOBEC-network reconstruction identifies pathways associated with survival in ovarian cancer.

BACKGROUND: Building up of pathway-/disease-relevant signatures provides a persuasive tool for understanding the functional relevance of gene alterations and gene network associations in multifactorial human diseases. Ovarian cancer is a highly complex heterogeneous malignancy in respect of tumor anatomy, tumor microenvironment including pro-/antitumor immunity and inflammation; still, it is generally treated as single disease. Thus, further approaches to investigate novel aspects of ovarian cancer pathogenesis aiming to provide a personalized strategy to clinical decision making are of high priority. Herein we assessed the contribution of the AID/APOBEC family and their associated genes given the remarkable ability of AID and APOBECs to edit DNA/RNA, and as such, providing tools for genetic and epigenetic alterations potentially leading to reprogramming of tumor cells, stroma and immune cells. RESULTS: We structured the study by three consecutive analytical modules, which include the multigene-based expression profiling in a cohort of patients with primary serous ovarian cancer using a self-created AID/APOBEC-associated gene signature, building up of multivariable survival models with high predictive accuracy and nomination of top-ranked candidate/target genes according to their prognostic impact, and systems biology-based reconstruction of the AID/APOBEC-driven disease-relevant mechanisms using transcriptomics data from ovarian cancer samples. We demonstrated that inclusion of the AID/APOBEC signature-based variables significantly improves the clinicopathological variables-based survival prognostication allowing significant patient stratification. Furthermore, several of the profiling-derived variables such as ID3, PTPRC/CD45, AID, APOBEC3G, and ID2 exceed the prognostic impact of some clinicopathological variables. We next extended the signature-/modeling-based knowledge by extracting top genes co-regulated with target molecules in ovarian cancer tissues and dissected potential networks/pathways/regulators contributing to pathomechanisms. We thereby revealed that the AID/APOBEC-related network in ovarian cancer is particularly associated with remodeling/fibrotic pathways, altered immune response, and autoimmune disorders with inflammatory background. CONCLUSIONS: The herein study is, to our knowledge, the first one linking expression of entire AID/APOBECs and interacting genes with clinical outcome with respect to survival of cancer patients. Overall, data propose a novel AID/APOBEC-derived survival model for patient risk assessment and reconstitute mapping to molecular pathways. The established study algorithm can be applied further for any biologically relevant signature and any type of diseased tissue.

Decreased melatonin levels and increased levels of advanced oxidation protein products in the seminal plasma are related to male infertility.

Melatonin, an indolamine secreted by the pineal gland, is known as a powerful free-radical scavenger and wide-spectrum antioxidant. Therefore, the aim of this study was to correlate markers of oxidative protein damage (advanced oxidation protein products, AOPPs) and the total antioxidant capacity (TAC) with melatonin levels in the seminal plasma of men with azoospermia (n=37), theratozoospermia (n=29) and fertile controls (normozoospermia, n=37). Melatonin concentration was measured by radioimmunoassay. The levels of AOPP as well as TAC efficiency (determined by the ferric reducing antioxidant power, FRAP) were estimated by spectrophotometric methods. The concentration of melatonin and AOPP significantly differed in azoospermic (P<0.0001) and theratozoospermic (P<0.0001) patients versus fertile men, and correlated negatively (r=-0.33, P=0.0016). The TAC levels were significantly higher in azoospermia than in theratozoospermia (P=0.0022) and the control group (P=0.00016). In azoospermia, the AOPP concentration was also significantly higher than that observed in theratozoospermia (P=0.00029). Decreased levels of melatonin together with elevated AOPP altered the oxidative-antioxidative balance in the ejaculate, thereby reducing fertility. Therefore, melatonin and AOPP levels may serve as additional diagnostic markers of semen quality and male reproductive potential.

Synergism of cyclin-dependent kinase inhibitors with camptothecin derivatives in small cell lung cancer cell lines.

Advanced small cell lung cancer (SCLC) has a dismal prognosis. Modulation of the camptothecin topotecan, approved for second-line therapy, may improve response. Our recent finding of synergistic enhancement of the cytotoxic activity of camptothecin (CPT) by cyclin-dependent kinase 4 inhibitors is extended here to a panel of camptothecin analogs comprising 10-hydroxy-CPT (HOCPT), topotecan (TPT; 9-[(dimethylamino)-methyl]-10-hydroxy-CPT), 9-amino-CPT (9AC), 9-nitrocamptothecin (rubitecan), SN38 (7-ethyl-10-hydroxycamptothecin) and 10-hydroxy-9-nitrocamptothecin (CPT109) in combination with PD0332991, CDK4I, roscovitine and olomoucine. SCLC cell lines employed are chemoresistant NCI-H417 and DMS153 and the chemosensitive SCLC26A line established at our institution. The CPT analogs exhibiting highest cytotoxicity towards the three SCLC lines tested were SN38 and 9AC, followed by rubitecan, HOCPT, TPT and CPT109. NCI-H417 and DMS153 revealed an approximately 25-fold and 7-fold higher resistance compared to the chemosensitive SCLC26A cell line. Whereas the CDK4/6 inhibitor PD0332991 proved less effective to chemosensitize SCLC cells to CPT analogs, the CDK inhibitors CDK4I, roscovitine and olomoucine gave comparable chemosensitization effects in combination with 9AC, SN38, rubitecan and to a lesser extent with TPT and CPT109, not directly related with topoisomerase mRNA expression. In conclusion, small chemical modifications of the parent CPT structure result in differing cytotoxicities and chemomodulatory effects in combination with CDKIs of the resulting analogs.

Automated REcognition of tissue-associated erythrocytes (ARETE)-a new tool in tissue cytometry.

Automated microscopic image analysis of immunofluorescence-stained targets on tissue sections is challenged by autofluorescent elements such as erythrocytes, which might interfere with target segmentation and quantification. Therefore, we developed an automated system (Automated REcognition of Tissue-associated Erythrocytes; ARETE) for in silico exclusion of erythrocytes. To detect erythrocytes in transmission images, a cascade of boosted decision trees of Haar-like features was trained on 8,640/4,000 areas (15 × 15 pixels) with/without erythrocytes from images of placental sections (4 µm). Ground truth data were generated on 28 transmission images. At least two human experts labelled the area covered by erythrocytes. For validation, output masks of human experts and ARETE were compared pixel-wise against a mask obtained from majority voting of human experts. F1 score, specificity, and Cohen's κ coefficients were calculated. To study the influence of erythrocyte-derived autofluorescence, we investigated the expression levels of a protein (receptor for advanced glycated end products; RAGE) in placenta and number of Ki-67-positive/cytokeratin 8-positive epithelial cells in colon sections. ARETE exhibited high sensitivity (99.87%) and specificity (99.81%) on a training-subset and processed transmission images (1,392 × 1,024 pixels) within 4 sec. ARETE and human expert's F1-scores were 0.55 versus 0.76, specificities 0.85 versus 0.92 and Cohen's κ coefficients 0.41 versus 0.68. A ranking of Cohen's κ coefficient by the scale of Fleiss certified "good agreement" between ARETE and the human experts. Applying ARETE, we demonstrated 4-14% false-positive RAGE-expression in placenta, and 18% falsely detected proliferative epithelial cells in colon, caused by erythrocyte-autofluorescence. ARETE is a fast system for in silico reduction of erythrocytes, which improves automated image analysis in research and diagnostic pathology.

Prognostic impact of tumor infiltrating CD8+ T cells in association with cell proliferation in ovarian cancer patients--a study of the OVCAD consortium.

BACKGROUND: Epithelial ovarian cancer is one of the most lethal gynecologic malignancies. Clinicopathological factors do not permit precise prognosis and cannot provide guidance to specific treatments. In this study we assessed tumor infiltrating CD8+ T cells in association with Ki67 proliferation index and evaluated their prognostic impact in EOC samples. METHODS: CD8+ cells and Ki67 proliferation index were immunohistochemically determined on tissue microarrays including 203 primary epithelial ovarian tumors. Additionally, CD8 gene expression was assessed with RT-qPCR. Correlations were analyzed using Pearson's correlation coefficients, ANOVA or T-test, or Fischer's exact tests. Prognostic impact was evaluated using the Kaplan-Meier method and Cox regression model. RESULTS: The density of CD8+ infiltrating lymphocytes did not correlate with tumor cell proliferation. Epithelial ovarian cancer patients with no Ki67+ cells in the tumor had a more than three times higher risk to die compared to the population with Ki67+ cells in the tumor (Hazard ratio (HR) = 3.34, 95%CI 1.59-7.04). High CD8+ cell infiltration was associated with improved overall survival (HR = 0.82, 95%CI 0.73-0.92). CONCLUSIONS: The density of tumor infiltrating lymphocytes is independent of tumor cell proliferation. Ovarian cancer patients with Ki67- tumors showed a significantly reduced overall survival, presumably due to no or poor response to platinum-based chemotherapy. Moreover, the association of high densities of tumor infiltrating cytotoxic T lymphocytes with a better overall survival was confirmed.

SLCO4A1 expression is associated with activated inflammatory pathways in high-grade serous ovarian cancer.

Patients with high-grade serous ovarian cancer (HGSOC) have a very poor overall survival. Current therapeutic approaches do not bring benefit to all patients. Although genetic alterations and molecular mechanisms are well characterized, the molecular pathological conditions are poorly investigated. Solute carrier organic anion transporter family member 4A1 (SLCO4A1) encodes OATP4A1, which is an uptake membrane transporter of metabolic products. Its expression may influence various signaling pathways associated with the molecular pathophysiological conditions of HGSOC and consequently tumor progression. RNA sequencing of 33 patient-derived HGSOC cell lines showed that SLCO4A1 expression was diverse by individual tumors, which was further confirmed by RT-qPCR, Western blotting and immunohistochemistry. Gene Set Enrichment Analysis revealed that higher SLCO4A1 level was associated with inflammation-associated pathways including NOD-like receptor, adipocytokine, TALL1, CD40, NF-κB, and TNF-receptor 2 signaling cascades, while low SLCO4A1 expression was associated with the mitochondrial electron transport chain pathway. The overall gene expression pattern in all cell lines was specific to each patient and remained largely unchanged during tumor progression. In addition, genes encoding ABCC3 along with SLCO4A1-antisense RNA 1, were associated with higher expression of the SLCO4A1, indicating their possible involvement in inflammation-associated pathways that are downstream to the prostaglandin E2/cAMP axis. Taken together, increased SLCO4A1/OATP4A1 expression is associated with the upregulation of specific inflammatory pathways, while the decreased level is associated with mitochondrial dysfunction. These molecular pathophysiological conditions are tumor specific and should be taken into consideration by the development of therapies against HGSOC.

Melatonin receptors, melatonin metabolizing enzymes and cyclin D1 in human breast cancer.

BACKGROUND: Melatonin suppresses breast cancer cell proliferation by inhibiting the upregulation of estrogen-induced cyclin D1 via its G-protein-coupled receptor MT1. Additionally, melatonin stimulates the expression of the estrogen sulfotransferase, SULT1E1. However, metabolism of melatonin via 6-hydroxylation by CYP1A1/1A2 and subsequent sulfonation by SULT1A1/1A3 decreases its intracellular concentration. This could have a negative impact on its oncostatic action in breast cancer. PATIENTS AND METHODS: In this pilot study, we performed immunohistochemical (IHC) analysis of MT1 and cyclin D1 in breast cancer specimens from 33 patients. Also, we investigated the expression of CYP1A1/1A2, SULT1A1/1A3/1E1,and cyclin D1 in cancer (CANC) and adjacent non-cancer (NCANC) specimens from 10 representative breast cancer patients using quantitative real-time reverse transcription polymerase chain reaction. RESULTS: CYP1A1-mRNA-expression was found only in three CANC and in one NCANC. CYP1A2 mRNA was below the detection limit in all patients. SULT1A1 was observed only in two of the 10 CANC and one of the 10 NCANC specimens. But, all 10 CANC and NCANC samples showed high SULT1A3 levels. Cyclin D1 mRNA levels were found in all 10 CANC and NCANC specimens. Furthermore, IHC-staining of cyclin D1 was observed in 27 of 33 CANC and correlated positively with estrogen receptor positivity (p = 0.015). CONCLUSION: The low or even absent expression of CYP1A1 or CYP1A2 in breast cancer specimens suggested that melatonin might be involved in cell cycle arrest.

Microarray analysis identifies distinct gene expression profiles associated with histological subtype in human osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumour. Currently osteosarcoma classification is based on histological appearance. It was the aim of this study to use a more systematic approach to osteosarcoma classification based on gene expression analysis and to identify subtype specific differentially expressed genes. We analysed the global gene expression profiles of ten osteosarcoma samples using Affymetrix U133A arrays (five osteoblastic and five non-osteoblastic osteosarcoma patients). Differential gene expression analysis yielded 75 genes up-regulated and 97 genes down-regulated in osteoblastic versus non-osteoblastic osteosarcoma samples, respectively. These included genes involved in cell growth, chemotherapy resistance, angiogenesis, steroid- and neuropeptide hormone receptor activity, acute-phase response and serotonin receptor activity and members of the Wnt/ß-catenin pathway and many others. Furthermore, we validated the highly differential expression of six genes including angiopoietin 1, IGFBP3, ferredoxin 1, BMP, decorin, and fibulin 1 in osteoblastic osteosarcoma relative to non-osteoblastic osteosarcoma. Our results show the utility of gene expression analysis to study osteosarcoma subtypes, and we identified several genes that may play a role as potential therapeutic targets in the future.

Actaea racemosa L. extract inhibits steroid sulfation in human breast cancer cells: Effects on androgen formation.

BACKGROUND: Actaea racemosa L., also known as black cohosh, is a popular herb commonly used for the treatment of menopausal symptoms. Because of its purported estrogenic activity, black cohosh root extract (BCE) may trigger breast cancer growth. STUDY DESIGN/METHODS: The potential effects of standardized BCE and its main constituent actein on cellular growth rates and steroid hormone metabolism were investigated in estrogen receptor alpha positive (ERα+) MCF-7 and -negative (ERα-) MDA-MB-231 human breast cancer cells. Cell numbers were determined following incubation of both cell lines with the steroid hormone precursors dehydroepiandrosterone (DHEA) and estrone (E1) for 48 h, in the presence and absence of BCE or actein. Using a validated liquid chromatography-high resolution mass spectrometry assay, cell culture supernatants were simultaneously analyzed for the ten main steroids of the estrogen pathway. RESULTS: Inhibition of MCF-7 and MDA-MB-231 cell growth (up to 36.9%) was observed following treatment with BCE (1-25 µg/ml) or actein (1-50 µM). Incubation of MCF-7, but not of MDA-MB-231 cells, with DHEA and BCE caused a 20.9% reduction in DHEA-3-O-sulfate (DHEA-S) formation, leading to a concomitant increase in the androgens 4-androstene-3,17-dione (AD) and testosterone (T). Actein was shown to exert an even stronger inhibitory effect on DHEA-S formation in MCF-7 cells (up to 89.6%) and consequently resulted in 12- to 15-fold higher androgen levels compared with BCE. The formation of 17ß-estradiol (E2) and its glucuronidated and sulfated metabolites was not affected by BCE or actein after incubation with the estrogen precursor estrone (E1) in either cell line. CONCLUSIONS: The results of the present study demonstrated that actein and BCE do not promote breast cancer cell growth or influence estrogen levels. However, androgen formation was strongly stimulated by BCE and actein, which may contribute to their ameliorating effects on menopausal symptoms in women. Future studies monitoring the levels of AD and T upon BCE supplementation of patients are warranted to verify an association between BCE and endogenous androgen metabolism.

Two common polymorphic variants of OATP4A1 as potential risk factors for colorectal cancer.

Genetic variations in the organic-anion-transporting polypeptide (OATP)-encoding solute carrier of organic anions (SLCO) genes can promote cancer development and progression. The overexpression of solute carrier organic anion transporter family member 4A1 (OATP4A1), a transporter for steroid hormones, prostaglandins, and bile acids, has been previously associated with tumor recurrence and progression in colorectal cancer (CRC). Therefore, the present study aimed to investigate the association between 2 frequent single nucleotide polymorphisms (SNPs) in SLCO4A1 (rs34419428, R70Q; rs1047099G, V78I) and CRC predisposition. Following restriction fragment length polymorphism-PCR analysis in 178 patients with CRC [Union for International Cancer Control (UICC) stage I/II] and 65 healthy controls, no significant difference was observed in allele frequency and the number of heterozygous/homozygous individuals between the groups. Notably, the R70Q minor allele was identified to be associated with the V78I minor allele in the genome. Comparing of the individual genotypes of CRC patients to clinical data, including sex, UICC-stage and relapse revealed no increased risk for CRC. In addition, the OATP4A1 immunoreactivity assay in paraffin-embedded CRC and adjacent non-tumorous mucosa sections, examined using quantitative microscopy image analysis, did not reveal any association with these polymorphisms. No significant differences were observed in the expression levels, localization, and sodium fluorescein transport capacity among the OATP4A1 variants, which was studied using functional assays in Sf9-insect and A431 tumor cells overexpressing the 2 single and a double mutant OATP4A1 SNP variants. These results suggested that the 2 most frequent polymorphisms located in the first intracellular loop of OATP4A1 do not associate with CRC predisposition and tumor recurrence. They are unlikely to affect the outcome of CRC in patients.

Metabolism of Estrogens: Turnover Differs Between Platinum-Sensitive and -Resistant High-Grade Serous Ovarian Cancer Cells.

High-grade serous ovarian cancer (HGSOC) is currently treated with cytoreductive surgery and platinum-based chemotherapy. The majority of patients show a primary response; however, many rapidly develop drug resistance. Antiestrogens have been studied as low toxic treatment options for HGSOC, with higher response rates in platinum-sensitive cases. Mechanisms for this difference in response remain unknown. Therefore, the present study investigated the impact of platinum resistance on steroid metabolism in six established HGSOC cell lines sensitive and resistant against carboplatin using a high-resolution mass spectrometry assay to simultaneously quantify the ten main steroids of the estrogenic metabolic pathway. An up to 60-fold higher formation of steroid hormones and their sulfated or glucuronidated metabolites was observed in carboplatin-sensitive cells, which was reversible by treatment with interleukin-6 (IL-6). Conversely, treatment of carboplatin-resistant cells expressing high levels of endogenous IL-6 with the monoclonal anti-IL-6R antibody tocilizumab changed their status to "platinum-sensitive", exhibiting a decreased IC50 value for carboplatin, decreased growth, and significantly higher estrogen metabolism. Analysis of these metabolic differences could help to detect platinum resistance in HGSOC patients earlier, thereby allowing more efficient interventions.

MCP-1 and MIP3-alpha serum levels in acute liver failure and molecular adsorbent recirculating system (MARS) treatment: a pilot study.

OBJECTIVE: The CC chemokines monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-3 alpha (MIP3-alpha) may be involved in the pathogenesis of acute liver failure (ALF) and acute-on-chronic liver failure (ACLF). In ALF and ACLF, the molecular adsorbent recirculating system (MARS) has been used to support liver function. Enhancement of MCP-1, as seen in other extracorporeal support systems such as haemodialysis, might thus have mitigated the beneficial effects of the MARS system in acute hepatic failure. MATERIAL AND METHODS: Serum concentrations of MCP-1 and MIP3-alpha were measured in 10 patients with ALF or ACLF treated with MARS. Thirteen patients suffering from chronic hepatic failure (CHF) and 15 healthy individuals served as controls. RESULTS: Baseline MCP-1 serum concentrations were significantly increased in ALF and ACLF patients as compared to patients with CHF (p=0.0027 and p=0.0046, respectively) and controls (p=0.0006 and p=0.0012, respectively). MIP3-alpha serum concentrations were also significantly enhanced in the ALF and ACLF groups as compared with those in CHF patients (p=0.0002 and p=0.0003, respectively) and controls (p<0.0001 and p<0.0001, respectively). Moreover, MIP3-alpha levels were significantly increased in CHF patients as compared to controls (p=0.0002). MCP-1 and MIP3-alpha concentrations did not change significantly during MARS treatment in ALF and ACLF patients. CONCLUSIONS: The CC chemokines MCP-1 and MIP3-alpha are increased in ALF and ACLF patients. MARS had no effect on MCP-1 and MIP3-alpha serum concentrations in patients with ALF and ACLF, and yielded no evidence of any harmful effects of the increase of these potentially hepatocidal chemokines.

Aqueous extracts of Cimicifuga racemosa and phenolcarboxylic constituents inhibit production of proinflammatory cytokines in LPS-stimulated human whole blood.

Cimicifuga racemosa (black cohosh) is commonly used in traditional medicines as treatment for menopausal symptoms and as an antiinflammatory remedy. To clarify the mechanism of action and active principle for the antiinflammatory action, the effects of aqueous C. racemosa root extracts (CRE) and its major constituents on the release of the proinflammatory cytokines IL-6, TNF-alpha, IFN-gamma, and the chemokine IL-8 were investigated in lipopolysaccharide (LPS)-stimulated whole blood of healthy volunteers. CRE (3 microg/microL and 6 microg/microL) reduced LPS-induced release of IL-6 and TNF-alpha in a concentration- and time-dependent manner and almost completely blocked release of IFN-gamma into the plasma supernatant. Except for IFN-gamma, these effects were attenuated at longer incubation periods. IL-8 secretion was stimulated by CRE. As shown by quantitative real-time RT-PCR, effects on cytokines were based on preceding changes in mRNA levels except for IL-8. According to their content in CRE, the phenolcarboxylic compounds caffeic acid, ferulic acid, and isoferulic acid, as well as the triterpene glycosides 23-epi-26-deoxyactein and cimigenol-3-O-xyloside, were tested at representative concentrations. Among these, isoferulic acid was the prominent active principle in CRE, responsible for the observed inhibition of IL-6, TNF-alpha, and IFN-gamma, but not for IL-8 stimulation. The effect of this compound may explain the antiinflammatory activities of CRE and its beneficial actions in rheumatism and other inflammatory diseases.

Antitumor activity of resveratrol and its sulfated metabolites against human breast cancer cells.

Resveratrol (3,4',5-trihydroxy- trans-stilbene) is a naturally occurring polyphenolic compound found in grapes, wine and medicinal plants with a variety of biological and pharmacological activities including pronounced anticancer properties. These effects are observed despite its extremely low bioavailability and rapid clearance from the circulation due to extensive sulfation and glucuronidation in the intestine and liver. In order to determine whether its metabolites demonstrate any cytotoxic properties, three major human sulfated conjugates of resveratrol were synthesized and their anticancer activity evaluated against three breast cancer cell lines (two hormone-dependent: MCF-7 and ZR-75-1; one hormone-independent: MDA-MB-231) and one immortalized breast epithelial cell line (MCF-10A). We found that, in contrast to resveratrol, all three sulfated metabolites were less potent against MCF-7, MDA-MB-231 and ZR-75-1 cells ( trans-resveratrol 3- O-sulfate < trans-resveratrol 4'- O-sulfate < trans-resveratrol 3- O-4'- O-disulfate) indicating that any conjugation of the phenolic groups with sulfuric acid strongly affecting the cytotoxicity. Interestingly, all sulfated metabolites were reduced about 10-fold, but showed nearly equal cytotoxicity towards nonmalignant MCF-10A breast cells (IC (50 s): 202-228 microM). In summary, in contrast to resveratrol its sulfated metabolites showed poor cytotoxicity in human malignant and nonmalignant breast cancer cell lines. However, the in vitro activity of the metabolites may not necessarily reflect their in vivo function, given the fact that the ubiquitously existing human sulfatases could convert the metabolites back to resveratrol in humans.

Resveratrol and other dietary polyphenols are inhibitors of estrogen metabolism in human breast cancer cells.

Polyphenols in foods and dietary supplements are commonly used for the prevention and treatment of a variety of malignancies, including breast cancer. However, daily intake by patients with breast cancer is controversial, as these compounds may stimulate cancer growth. Estrogens serve key roles in breast cancer cell proliferation; therefore, understanding the interaction between endogenous steroid hormones and natural dietary polyphenols is essential. Currently, comprehensive knowledge regarding these effects remains limited. The current review summarizes the dose-dependent in vitro and in vivo interactions of resveratrol and other dietary polyphenols with estrogen precursors, active estrogens, catechol estrogens and their respective glucuronidated, sulfated, glutathionated or O-methylated metabolites in estrogen receptor alpha negative (ERα-) and positive (ERα+) breast cancer. Which estrogen-metabolizing enzymes are affected by polyphenols is also reviewed in detail. Furthermore, the impacts of dose and therapy duration on disease development and progression in patients with breast cancer are discussed. The present article is part of a Special Issue titled 'CSR 2018'.

Abundance of the Organic Anion-transporting Polypeptide OATP4A1 in Early-Stage Colorectal Cancer Patients: Association With Disease Relapse.

The abundance of OATP4A1 in colorectal cancer (CRC) might be related to tumor progression. This was studied by immunohistochemistry on paraffin-embedded samples obtained from 178 patients (43 patients with a relapse within 5 y) with early-stage CRC. Positivity for OATP4A1 in tumor cells and noncancerous mucosal cells was proved by double-immunofluorescence staining with antibodies against OATP4A1 and keratin 8, whereas antibodies against appropriate CD markers were used to identify immune cells. Automated microscopic image analysis was used to measure the percentage of OATP4A1-positive cells and OATP4A1 staining intensity in tumor, immune, and adjacent normal-looking mucosal cells separately, as well as in the mucosal and immune cells of 14 nonmalignant tissue samples. In CRC the percentage of OATP4A1-positive cells, but not staining intensity, was significantly higher in tumor and mucosal cells adjacent to the tumor compared to the mucosa of nonmalignant samples (P<0.001 each). No difference was registered between immune cells in malignant and nonmalignant samples. Importantly, high levels of OATP4A1 in immune (odds ratio, 0.73; confidence interval, 0.63-0.85; P<0.001), and tumor cells (odds ratio, 0.79; confidence interval, 0.69-0.91; P<0.001) are significantly associated with a low risk of recurrence and also significantly enhance the discriminative power of other clinical parameters [such as International Union Against Cancer (UICC), adjuvant therapy, localization of the primary tumor] of the risk of relapse (receiver operating characteristics analysis; P=0.002). Using an advanced digital microscopic quantification procedure, we showed that OATP4A1 abundance is negatively associated with tumor recurrence in early-stage CRC. This digital scoring procedure may serve as a novel tool for the assessment of potential prognostic markers in early-stage CRC.

Metabolism of resveratrol in breast cancer cell lines: impact of sulfotransferase 1A1 expression on cell growth inhibition.

Resveratrol is a polyphenolic compound present in grapes and wine with anticancer activities that undergoes pronounced metabolism in humans. In order to determine whether metabolism of resveratrol also occurs in tumor cells and whether biotransformation has any impact on cytotoxicity, metabolism experiments were conducted with hormone-dependent ZR-75-1 and hormone-independent MB-MDA-231 human breast cancer cells. Along with resveratrol, it was possible to identify one metabolite, namely, resveratrol-3-O-sulfate in both cell lines. Its concentration in the cytoplasm and culture medium was 5.4- to 9-fold higher in ZR-75-1 cells than in MDA-MB-231 cells, concomitant with a 3.1-fold higher IC(50) value in the ZR-75-1 cell line (74 microM compared to 38 microM). By using RT-PCR, expression of sulfotransferase (SULT)1A1 mRNA, but not of other SULTs investigated, showed a close correlation with resveratrol 3-O-sulfate formation which was particularly high in ZR-75-1 and very low in MDA-MD-231 cells. In conclusion, we demonstrate that SULT1A1-based biotransformation reduces the anticancer activity of resveratrol in breast cancer cells, which must be considered in humans following oral uptake of dietary resveratrol as a chemopreventive agent.

Cell cycle dysregulation influences survival in high risk breast cancer patients.

BACKGROUND: Cell cycle progression is regulated by cyclin dependent kinases (cdk) and cdk inhibitors. Recent immunohistological studies suggested that dysregulation of cyclin A, cyclin D, cyclin E, p16(ink4), p21(waf1/cip1), and p27(kip1) are of prognostic value in patients with breast cancer. Our study represents the first comprehensive immunohistochemical cell cycle marker analysis for cdc25A, cyclin A, cyclin D, cyclin E, p16(ink4), p21(waf1/cip1), p27(kip1), and pRb in tumor tissue and adjacent benign breast tissue from 69 primarily untreated breast cancer patients. METHODS: Immunhistochemistry using primary monoclonal antibodies to detect cdc 25A, cyclin A, cyclin D, cyclin E, p16(ink4), p21(waf1/cip1), p27(kip1), and pRb has been performed. RESULTS: Sixty-nine patients with untreated, invasive breast cancer (n = 69) were divided into a low/ intermediate and a high risk group according to the St. Gallen 2005 consensus conference. High risk patients (n = 22) had a significantly (p = 0.003) shorter mean and median survival (282.85 weeks; 383.0 weeks, respectively) than low/intermediate risk patients (375.41 weeks; not reached yet, respectively). A subgroup of high risk breast cancer patients characterized in addition by overexpression of cdc25A, cyclin A, cyclin E, p16(ink4a), and p27(kip1) experienced a shortened mean survival of 222.03, 235.71, 257.25, 239.18, and 261.94 weeks, respectively. Regarding benign breast tissue adjacent to breast cancer tissue, 59.4% of the patients investigated overexpressed cdc25A, 23.2% overexpressed pRb, and 63.2% exerted dysregulation of p27(kip1) while they proved to be negative for immunohistochemical staining regarding all other markers tested. CONCLUSION: The immunohistological analyses of cdc25A, cyclin A, cyclin E, p16(ink4a), and p27(kip1) have the potential for further refining the risk assessment in patients with untreated breast cancer who belong to the high risk category defined according to the St. Gallen 2005 consensus conference. These cell cycle markers define a subgroup of high risk patients with even higher risk of metastazation and shortened survival. For confirmation a prospective study using standardized laboratory procedures in a larger population is needed.