Differential microRNA Expression Analysis in Patients with HPV-Infected Ovarian Neoplasms.

This study aimed to identify microRNAs (miRNAs) whose expression levels are altered by high-risk human papillomavirus (HR-HPV) infection in women with epithelial ovarian neoplasms. MiRNA expression was quantified by real-time polymerase chain reaction, while HR-HPV DNA was quantified using digital-droplet PCR. Analysis of 11 miRNAs demonstrated significantly lower hsa-miR-25-5p expression in HPV-infected compared to uninfected ovarian tissues (p = 0.0405), while differences in miRNA expression in corresponding serum were statistically insignificant. The expression of hsa-miR-218-5p in ovarian tumors was significantly higher in high-grade serous ovarian carcinoma (HGSOC) cases than in other neoplasms (p = 0.0166). In addition, hsa-miR-218-5p was significantly upregulated, whereas hsa-miR-191-5p was significantly downregulated in tissues with stage III/IV FIGO (p = 0.0009 and p = 0.0305, respectively). Using unsupervised clustering, we identified three unique patient groups with significantly varied frequencies of HPV16/18-positive samples and varied miRNA expression profiles. In multivariate analysis, high expression of hsa-miR-16-5p was an independent prognostic factor for poor overall survival (p = 0.0068). This preliminary analysis showed the changes in miRNA expression in ovarian neoplasms during HPV infection and those collected from HGSOCs or patients with advanced disease. This prospective study can provide new insights into the pathogenesis of ovarian neoplasms and host-virus interactions.

The Toll-like Receptor 4 Polymorphism Asp299Gly Is Associated with an Increased Risk of Ovarian Cancer.

Ovarian cancer (OC) is one of the most common cancers threatening women's lives around the world. Epithelial ovarian tumors represent the most common ovarian neoplasms. Most OC patients are diagnosed at the advanced stage, and there is an urgent need to identify novel biomarkers of the disease. Single-nucleotide polymorphisms (SNPs) in TLR genes may serve as crucial markers of cancer susceptibility. We investigated the frequency of TLR polymorphisms in a group of 200 women, including 70 with OC. Four SNPs, two each in TLR4 (rs4986790 and rs4986791) and TLR9 (rs187084 and rs5743836), were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The digested fragments were separated and identified by multicapillary electrophoresis. The load quantification of human papillomavirus (HPV) types 16/18 was determined using a digital droplet PCR method. We found an increased frequency of heterozygous genotype and minor allele of the TLR4 rs4986790 SNP in women with OC compared with healthy controls, and this result remained highly significant after Bonferroni's correction for multiple testing (p < 0.0001). No evidence of linkage disequilibrium was found with any of the examined TLR SNPs. The findings suggest that the TLR4 Asp299Gly polymorphism could be a genetic risk factor for the development of OC.

Stimulation of lactate receptor (HCAR1) affects cellular DNA repair capacity.

Numerous G-protein coupled receptors have been reported to enhance cancer cell survival and resistance to clinically used chemotherapeutics. Recently, hydroxycarboxylic acid receptor 1 (HCAR1) was shown to drive lactate-dependent enhancement of cell survival and metastasis in pancreatic and breast cancers. Furthermore, our previous study confirmed the involvement of HCAR1 in lactate-related enhancement of DNA repair in cervical cancer cells. In the present study, we examined the possible mechanisms of HCAR1-mediated enhancement of DNA repair capacity. We observed that the HCAR1 agonist dihydroxybenzoic acid (DHBA) up-regulated BRCA1 (breast cancer type 1 susceptibility protein) and NBS1 (Nijmegen breakage syndrome 1) expression in HeLa cells. Moreover, HCAR1 silencing decreased mRNA and protein levels of BRCA1 by 30% and 20%, respectively. Immunocytochemical analyses of BRCA1, nibrin and DNA-PKcs indicated an increased accumulation of these proteins in cell nuclei after DHBA stimulation. Subsequently, these changes in the DNA repair protein levels translated into an enhanced DNA repair rate after doxorubicin treatment, as shown by γ-H2AX and comet assay experiments. In contrast, the down-regulation of HCAR1 decreased the efficiency of DNA repair. Finally, we observed the abrogation of DHBA-driven BRCA1 protein up-regulation and enhanced DNA repair following the preincubation of cells with the PKC inhibitor Gö6983. Taken together, our data indicate that lactate receptor/HCAR1 expression in cervical carcinoma cells may contribute to the modulation of cellular DNA repair mechanisms.

Doxorubicin-transferrin conjugate triggers pro-oxidative disorders in solid tumor cells.

The formation of reactive oxygen species (ROS) is a widely accepted mechanism of doxorubicin (DOX) toxicity toward cancer cells. However, little is known about the potential of new systems, designed for more efficient and targeted doxorubicin delivery (i.e. protein conjugates, polymeric micelles, liposomes, monoclonal antibodies), to induce oxidative stress (OS) in tumors and hematological malignancies. Therefore, the objective of our study was to determine the relation between the toxicity of doxorubicin-transferring (DOX-TRF) conjugate and its capability to generate oxidative/nitrosative stress in solid tumor cells. Our research proves that DOX-TRF conjugate displays higher cytotoxicity towards lung adenocarcinoma epithelial (A549) and hepatocellular carcinoma (HepG2) cell lines than the reference free drug (DOX) and induces more extensive OS, characterized by a significant decrease in the total cellular antioxidant capacity, glutathione level and amount of -SH groups and an increase in hydroperoxide content. The intracellular redox imbalance was accompanied by changes in the transcription of genes encoding key antioxidant enzymes engaged in the sustaining of cellular redox homeostasis: superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST) and glutathione peroxidase (GP).

Toxicity of doxorubicin-transferrin conjugate is connected to the modulation of Wnt/ß-catenin pathway in human leukemia cells.

Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells caused by constitutive activation of the BCR/ABL tyrosine kinase. However, the tyrosine kinase inhibitors like imatinib mesylate are not effective in the patients with advanced-stage of CML. Hence, there is an urgent need for new approaches to overcome a cancer cell's resistance in CML long term therapy. Development of new drug carriers, is presently one of the most challenging tasks in experimental oncology. In this report we investigated whether the toxicity of newly synthetized doxorubicin transferrin conjugate (DOX-TRF) may be connected to the limitation of multidrug resistance in CML cells by the alternations of Wnt/ß-catenin signaling pathway. The studies were performed on human chronic myeloid leukemia cell lines sensitive (K562) and resistant (K562/DOX) to doxorubicin. Our research proves that DOX-TRF conjugate displays higher cytotoxicity toward both examined cell lines than the reference free drug (DOX) and induces more extensive pro-apoptotic changes. Moreover, by the of engagement of Wnt pathway agonist (LiCl) and antagonist (ICG 001) we demonstrate that DOX-TRF conjugate effectively reduces transcription of key genes involved in ß-catenin signaling transduction trial (Wnt3a, DVL-1, FZD-3, LRP5, ß-catenin, DKK2) and triggers morphology alternations of CML cells.

L- and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation.

BACKGROUND: The consideration of lactate as an active metabolite is a newly emerging and attractive concept. Recently, lactate has been reported to regulate gene transcription via the inhibition of histone deacetylases (HDACs) and survival of cancer cells via hydroxycarboxylic acid receptor 1 (HCAR1). This study examined the role of L- and D-lactate in the DNA damage response in cervical cancer cells. METHODS: Three cervical cancer cell lines were examined: HeLa, Ca Ski and C33A. The inhibitory activity of lactate on HDACs was analysed using Western blot and biochemical methods. The lactate-mediated stimulation of DNA repair and cellular resistance to neocarzinostatin, doxorubicin and cisplatin were studied using γ-H2AX, comet and clonogenic assays. HCAR1 and DNA repair gene expression was quantified by real-time PCR. DNA-PKcs activity and HCAR1 protein expression were evaluated via immunocytochemistry and Western blot, respectively. HCAR1 activation was investigated by measuring intracellular cAMP accumulation and Erk phosphorylation. HCAR1 expression was silenced using shRNA. RESULTS: L- and D-lactate inhibited HDACs, induced histone H3 and H4 hyperacetylation, and decreased chromatin compactness in HeLa cells. Treating cells with lactate increased LIG4, NBS1, and APTX expression by nearly 2-fold and enhanced DNA-PKcs activity. Based on γ-H2AX and comet assays, incubation of cells in lactate-containing medium increased the DNA repair rate. Furthermore, clonogenic assays demonstrated that lactate mediates cellular resistance to clinically used chemotherapeutics. Western blot and immunocytochemistry showed that all studied cell lines express HCAR1 on the cellular surface. Inhibiting HCAR1 function via pertussis toxin pretreatment partially abolished the effects of lactate on DNA repair. Down-regulating HCAR1 decreased the efficiency of DNA repair, abolished the cellular response to L-lactate and decreased the effect of D-lactate. Moreover, HCAR1 shRNA-expressing cells produced significantly lower mRNA levels of monocarboxylate transporter 4. Finally, the enhancement of DNA repair and cell survival by lactate was suppressed by pharmacologically inhibiting monocarboxylate transporters using the inhibitor α-cyano-4-hydroxycinnamic acid (α-CHCA). CONCLUSIONS: Our data indicate that L- and D-lactate present in the uterine cervix may participate in the modulation of cellular DNA damage repair processes and in the resistance of cervical carcinoma cells to anticancer therapy.

Molecular damage caused by generation of reactive oxygen species in the redox cycle of doxorubicin-transferrin conjugate in human leukemia cell lines.

In this study we focused on evaluation of the pro-oxidant properties of doxorubicin-transferrin (DOX-TRF) conjugate and its potency to damage macromolecules which are components of cellular compartments. Our experiments were performed on two human leukemia cell lines: K562 (chronic erythromyeloblastoid leukemia) and CCRF-CEM (acute lymphoblastic leukemia). We determined the reactive oxygen species (ROS) production and programmed cell death (PCD) induction by free DOX and its conjugate. Besides this, the lipid peroxidation and protein damage which can be provoked by DOX alone and DOX-TRF conjugate were assessed. ROS were produced in leukemia cells incubated with free DOX and DOX-TRF conjugate and the extent of apoptosis and necrosis was strongly dependent on the cell line, sensitivity to drug and time of incubation with the investigated compounds. The role of ROS in DOX-TRF conjugate-induced cell death was confirmed by the diminution effects of the antioxidant vitamin C.

Relationship between therapeutic efficacy of doxorubicin-transferrin conjugate and expression of P-glycoprotein in chronic erythromyeloblastoid leukemia cells sensitive and resistant to doxorubicin.

BACKGROUND: Conjugation of anti-neoplastic agents with human proteins is a strategy to diminish the toxic side effects of anthracycline antibiotics. We have developed a novel doxorubicin-transferrin (DOX-TRF) conjugate aimed to direct anticancer drugs against therapeutic targets that display altered levels of expression in malignant versus normal cells. Our previous work has shown that the cellular bio-distribution of the conjugate is dependent on a dynamic balance between influx and efflux processes. Here, we set out to investigate whether P-glycoprotein (P-gp) expression may affect DOX-TRF conjugate-induced cellular drug accumulation and cytotoxicity. RESULTS: All experiments were carried out on human erythromyeloblastoid cells exhibiting P-gp over-expression (K562/DOX) and its drug sensitive parental line (K562). MTT cytotoxicity, flow cytometry, fluorescence microscopy and RT-PCR assessments revealed that the investigated conjugate (DOX-TRF) possesses a greater cytotoxic potential than free DOX. CONCLUSION: Our data suggest that the newly developed DOX-TRF conjugate is a less P-gp dependent substrate than free DOX and, consequently, may be used in a clinical setting to increase treatment efficacy in resistant human tumors.

Changes in the activity of antioxidant barrier after treatment of K562 and CCRF-CEM cell lines with doxorubicin-transferrin conjugate.

Doxorubicin (DOX), one of the oldest member of the anthracycline antibiotics, has been administered for over 50 years to patients with leukemias and solid tumors. However, the high unspecified DOX toxicity, related to reactive oxygen species (ROS), affects its limitation in clinical application. Therefore we proposed the usage of human transferrin as a doxorubicin carrier in order to improve the quality of doxorubicin application in conventional chemotherapy. In this study we continue our investigations related to the mechanism of the toxicity of doxorubicin-transferrin (DOX-TRF) conjugate in human leukemia cells. Consequently, we are now concentrating on the influence of this compound on the antioxidative system in K562 and CCRF-CEM cell lines (chronic erythromyeloblastoid leukemia and acute lymphoblastic leukemia cells, respectively). We carried out a neutral red cytotoxicity assay, reduced (GSH) and total (GSH + GSSG) glutathione content, alterations in the activity of catalase and enzymes responsible for maintaining glutathione in reduced form. Exposure of leukemia cells to the investigated anticancer agents caused a time-dependent depletion of intracellular GSH, accompanied by an increase of catalase activity. Moreover, analysis of GSH-related enzymes showed a significant increase in the activities of thioredoxin reductase and glutathione peroxidase after DOX-TRF application. In contrast, glutathione reductase activity was reduced by conjugate treatment to 50%. Significant differences between the pro-oxidative actions of the investigated anticancer compounds were observed in RT-PCR experiments, which confirmed that changes in the activity of catalase and GSH-related enzymes are strictly correlated with their gene transcription changes.

Beta amyloid effects on expression of multidrug efflux transporters in brain endothelial cells.

ABC (ATP Binding Cassette) efflux transporters at the blood-brain barrier, P-glycoprotein (ABCB1), multidrug resistance associated protein 4 (ABCC4) and breast cancer resistance protein (ABCG2), are important for protecting the brain from circulating xenobiotics. Their expression is regulated by signals from surrounding brain tissue that may alter in CNS pathologies. Differences have been reported in transporter expression on brain vasculature of Alzheimer's subjects where raised levels of ß-amyloid (Aß) occur. The present study examines in vitro the effects of Aß using immortalised brain endothelial cells (hCMEC/D3). Significantly lower expression of ABCB1 but not ABCC4 or ABCG2 was found following exposure to Aß(1-42) peptide but not its scrambled equivalent. This was evident at both protein and transcript level and was reflected in lower transcriptional activity of the ABCB1 promoter as judged from the luciferase reporter gene assay and in decreases in ABCB1-mediated efflux of rhodamine 123. Aß exposure also affected Wnt/ß-catenin signalling, decreasing levels of ß-catenin protein, reducing activation of TOPFLASH and increasing transcript levels of endogenous inhibitor, Dkk-1. Application of Wnt3a reversed the Aß-induced changes to ABCB1 protein. These results suggest that Aß may impair Wnt/ß-catenin signalling at the blood-brain barrier but that activation of this pathway may restore ABCB1.

Nitric oxide contributes to hypoxia-reoxygenation-induced P-glycoprotein expression in rat brain endothelial cells.

Ischemia-reperfusion leads to increased levels at the blood-brain barrier of the multidrug efflux transporter, P-glycoprotein that provides protection to the brain by limiting access of unwanted substances. This is coincident with the production of nitric oxide. This present study using immortalized rat brain endothelial cells (GPNTs) examines whether following hypoxia-reoxygenation, nitric oxide contributes to the alterations in P-glycoprotein levels. After 6 h of hypoxia, both nitric oxide and reactive oxygen species, detected intracellularly using fluorescent monitoring dyes, were produced in the subsequent reoxygenation phase coincident with increased P-glycoprotein. The evidence that nitric oxide can directly affect P-glycoprotein expression was sought by applying S-nitroso-N-acetyl-DL: -penicillamine that as shown increased the nitric oxide generation. Sodium nitroprusside, though more effective at increasing P-glycoprotein expression, appeared to produce different reactive species. Real time RT-PCR analysis revealed the predominant form of nitric oxide synthase in these cells to be endothelial, inhibition of which partially prevented the increase in P-glycoprotein during reoxygenation. These data indicate that the production of nitric oxide by endothelial nitric oxide synthase during reoxygenation can influence P-glycoprotein expression in cells of the blood-rat brain barrier, highlighting another route by which nitric oxide may protect the brain.

P-glycoprotein expression in immortalised rat brain endothelial cells: comparisons following exogenously applied hydrogen peroxide and after hypoxia-reoxygenation.

Levels of multidrug efflux transporter P-glycoprotein (P-gp) on endothelial cells lining brain blood vessels are important for limiting access of many compounds to the brain. In vivo studies have indicated that ischaemia-reperfusion that generates reactive oxygen species also increases P-gp levels in brain endothelial cells. To investigate possible mechanisms, in vitro studies were performed on immortalised (GPNT) and primary rat brain endothelial cells. Exposure to hydrogen peroxide (200 microM) resulted in intracellular oxidative stress as detected from higher levels of dichlorofluorescein fluorescence and raised levels of P-gp protein, mdr1a and mdr1b transcripts and, in GPNT cells, increased mdr1a and mdr1b promoter activity. The P-gp protein increases were abolished by pre-treatment with polyethylene glycol-catalase and were curtailed by co-culture with primary rat astrocytes. Exposure of GPNT cells to 6 h hypoxia followed by 24 h reoxygenation produced less intracellular oxidative stress as judged from smaller increments in dichlorofluorescein fluorescence but still resulted in raised levels of P-gp protein, an effect partially abolished by pre-treatment with polyethylene glycol-catalase. However, transcript levels and promoter activities were not significantly increased. These data suggest that hydrogen peroxide contributes to P-gp up-regulation following hypoxia-reoxygenation but the underlying mechanisms of its actions differ from those occurring after direct hydrogen peroxide application.

Activation of beta-catenin signalling by GSK-3 inhibition increases p-glycoprotein expression in brain endothelial cells.

This study investigates involvement of beta-catenin signalling in regulation of p-glycoprotein (p-gp) expression in endothelial cells derived from brain vasculature. Pharmacological interventions that enhance or that block beta-catenin signalling were applied to primary rat brain endothelial cells and to immortalized human brain endothelial cells, hCMEC/D3, nuclear translocation of beta-catenin being determined by immunocytochemistry and by western blot analysis to confirm effectiveness of the manipulations. Using the specific glycogen synthase kinase-3 (GSK-3) inhibitor 6-bromoindirubin-3'-oxime enhanced beta-catenin and increased p-gp expression including activating the MDR1 promoter. These increases were accompanied by increases in p-gp-mediated efflux capability as observed from alterations in intracellular fluorescent calcein accumulation detected by flow cytometry. Similar increases in p-gp expression were noted with other GSK-3 inhibitors, i.e. 1-azakenpaullone or LiCl. Application of Wnt agonist [2-amino-4-(3,4-(methylenedioxy) benzylamino)-6-(3-methoxyphenyl)pyrimidine] also enhanced beta-catenin and increased transcript and protein levels of p-gp. By contrast, down-regulating the pathway using Dickkopf-1 or quercetin decreased p-gp expression. Similar changes were observed with multidrug resistance protein 4 and breast cancer resistance protein, both known to be present at the blood-brain barrier. These results suggest that regulation of p-gp and other multidrug efflux transporters in brain vasculature can be influenced by beta-catenin signalling.