Supplementary Figures S1-S2 from ABCC2 (MRP2, cMOAT) Can Be Localized in the Nuclear Membrane of Ovarian Carcinomas and Correlates with Resistance to Cisplatin and Clinical Outcome

Rhythmic masticatory muscle activity (RMMA) is a periodic muscle activity that characterises sleep bruxism (SB) events. These can occur as a single event, in pairs, or in clusters. Since RMMA episodes often occur in clusters and the relevance of this occurrence is unknown, we conducted a study to investigate the effect of RMMA clusters on sleep fragmentation and the severity of orofacial muscle pain. This study involved a secondary analysis using data from 184 adult subjects with orofacial muscle pain who underwent definitive polysomnography (PSG) for sleep bruxism diagnosis. Self-reported orofacial muscle pain (OFMP) was assessed using the numeric rating scale, and additional evaluation of side-to-side equivalence (symmetry) was described using a binary system. Among the 184 participants, 60.8% (n = 112) did not exhibit clusters and among the 72 participants with clusters, 36.1% (n = 26) and 63.9% (n = 46) were in the high and low RMMA frequency groups, respectively. The high SB group had significantly three times more phasic RMMA events than the noncluster group. A total of 89.67% (n = 165) of subjects reported orofacial muscle pain. While there was no difference in the severity of OFMP among groups, a significant decrease in symmetry between the severity of temporal muscle pain on the left and right sides was noted in the cluster group compared with the noncluster group. Clustering of RMMA events is associated with sleep fragmentation. The asymmetry of temporal muscle pain is related to the presence of RMMA clusters in sleep bruxism.

Platelet-rich plasma (PRP) is used in medicine as a source of autologous growth factors in different indications. At present, PRP is applied increasingly frequently in aesthetic medicine with the aim of skin revitalization. Until now, the mechanisms of PRP effects in healthy human skin treated with aesthetic goals have not been identified in detail. This study aimed to examine PRP effects on the synthesis of procollagen type I in human skin. This study was a prospective, single-center, single-dose, open-label, non-randomized controlled clinical study. The study was conducted on a group of 10 volunteers in whom forearm skin was injected with PRP, while the placebo control group received injections of 0.9% NaCl. Expression of procollagen type I was examined after 21 days using immunohistochemistry. The study demonstrated that skin fragments subjected therapy using PRP demonstrated a significantly higher expression of procollagen than that which was observed in placebo controls. The study demonstrated that PRP stimulated collagen expression in healthy human skin.

Procedures with polydioxanone (PDO) threads are increasingly used for aesthetic indications. To date, eight cases of serious complications following the use of PDO threads have been published. In this case report, we present a case of a serious bacterial complication after a procedure with four PDO threads. A 52-year-old female patient presented to our center 1 month after undergoing the procedure at another center. Despite early symptoms, no treatment had previously been implemented. Perforating abscesses were found along the course of the threads. After 5 days of antibiotic therapy (amoxicillin 875 mg and clavulanic acid 125 mg p.o. every 12 hours), the threads were surgically removed due to skin rippling. During the procedure, the threads were found to be fragile, and several incisions were necessary to remove them. After 1 month, no signs of inflammation were reported. However, persistent deformities occurred due to delayed treatment implementation. Bacterial complications seem to be a typical complication following the procedure with PDO threads. PDO threads can be difficult to remove due their fragility. The possible need for surgical removal of the threads should be considered when selecting areas for application.

Gliomas are a group of malignant brain tumors. Despite significant efforts to optimize treatment options for patients with high-grade glioma, the prognosis of the overwhelming majority of patients remain poor. This bleak prognosis despite treatment of the glioma, is partly due to the tendency of therapeutics to diffusely penetrate into the neighboring brain tissues, but also due to the innate resistance of these tumors to chemotherapy and radiation. Garlic contains water-soluble and oil-soluble sulfur compounds. The oil-soluble compounds, including diallyl sulfide, diallyl disulfide (DADS), diallyl trisulfide and ajoene, are more effective potential anti-cancer treatments than the water-soluble compounds. There are several studies examining the effects of oil-soluble compounds on various types of cancer cells, although, to the best of our knowledge, there are no studies examining the effects of these compounds on glioma cells. The aim of the present study was to investigate the potential anti-glioma properties of DAD and garlic oil on proliferation and induction of apoptosis in four different types of glioma cell lines representative of different grades of the disease. The results showed that garlic oil exhibits favorable anti-cancer potential towards gliomas of various degrees of differentiation.

Polo-like kinase 1 (PLK1) is a serine-threonine kinase that plays a crucial role in the regulation of cell division. In addition, it acts as a modulator of the DNA damage response and as a novel factor in the maintenance of genome stability during DNA replication. The present study aimed to reveal the associations between PLK1 expression and clinicopathological features of patients with breast cancer (BC), particularly patient survival at 5-, 10- and 15-year follow-up. PLK1 expression was evaluated immunohistochemically in routine diagnostic tissue specimens from 83 patients treated radically for stage II BC. Kaplan-Meier analysis revealed a correlation between PLK1 overexpression and long-term survival. High PLK1 immunoreactivity was associated with shorter cancer-specific overall survival (CSOS) and disease-free survival (P=0.00001 and 0.00013, respectively). Multivariate analysis confirmed the negative prognostic significance of PLK1 overexpression for CSOS in all 83 patients (P=0.00030). Furthermore, analogous correlations were observed in both subgroups with and without nodal metastases (P=0.01400 and 0.01200, respectively). The present results indicate that PLK1 expression has a prognostic role in early BC. Immunohistochemical assessment of PLK1 reactivity may potentially become a qualifier for inclusion of PLK1 inhibitor therapy.

Electropulse technology using nanosecond pulsed electric fields (nsPEFs) presents a new stimulating factor to interfere with cell functions or induce cell death in cancer cells. nsPEFs permeablilizes cells generating large numbers of non-stable nanopores in all cell membranes. This newly created pores can be efficiently used by drugs for a molecule transport in anticancer therapy. In the current research we attempted to apply nsPEF to transport strontium ranelate. Two human gastric cancer cell line (EPG85-257P and EPG85-257RDB), colon adenocarcinoma (LS-180), breast adenocarcinoma cell lines (MCF-7/WT and MCF-7/DX), melanoma (Me45), epidermal cancer (A431), normal keratinocytes (HaCaT) and macrophages (P388/D1) were used for nano-electrochemotherapy in vitro. The following electrical field parameters: EP - 12.5 kV/cm, 25 kV/cm, 37 kV/cm, with repetition frequency of 100 Hz, 200 impulses of 10 ns each, and time rise of 2 ns. MTT assay was applied for evaluation of combined therapy effectiveness after 24 and 72 hours. The intracellular calcium level was detected by Fluo-4, membrane permeabilization was detected by CellMask – membrane marker. The nsPEF-strontium therapy was significantly efficient in adenocarcinoma type of cells. Cells derived from skin were resistant to the applied therapy.

Introduction: Electroporation (EP) is commonly applied for effective drug transport thorough cell membranes based on the application of electromagnetic field. When applied with cytostatics, it is called electrochemotherapy (ECT) - a quite new method of cancer treatment. A high-voltage pulse causes the formation of temporary pores in the cell membrane which create an additional way for the intracellular drug transport. In the current work, EP was effectively merged with the already known photodynamic therapy (PDT) to selective photosensitizers' delivery to diseased tissue. The application of electroporation can reduce the dose of applied drug.Research objective: The aim of research was to evaluate the effectiveness of photodynamic reaction using two near infrared cyanines (AlPc and Pc green) combined with electroporation in two human gastric adenocarcinoma cell lines.Materials and methods: Two human cell lines - EPG85-257P (parental) and EPG85-257RDB (resistant to daunorubicin) - of gastric cancer were used. The effect of two photosensitizers (aluminum 1,8,15,22-tetrakis(-phenylthio)-29H,31H-phthalocyanine chloride and Phthalocyanine green) was investigated. The efficiency of EP parameters was assessed by propidium iodide uptake. The viability assay was applied to analyse EP, PDT and EP-PDT effect. Cyanine localization was determined by confocal microscopy. Immunocytochemical evaluation of manganese superoxide dismutase and glutathione S-transferase-pi was determined after applied therapies.Results: PDT in combination with EP affected the viability of EPG85-257P and EPG85-257RDB cells negatively while both cyanine were used. The most evident changes were observed in the following concentrations: 15, 10 and 5 mu M. The optimal field strength for enhanced EP-PDT was 800 and 1200 V/cm. AlPc distributed selectively in the lysosomes of parental cell line.Conclusions: PDT, enhanced by EP, caused decreased viability when compared to the application of PDT alone. Both phthalocyanines found to be more effective after electroporation. Due to the low concentration of light-sensitive compounds and safety of electroporation itself, a treatment plan can be an alternative therapeutic modality against gastric adenocarcinomas. (C) 2014 Elsevier Masson SAS. All rights reserved.

OBJECTIVE:Numerous studies suggest that cyclooxygenase-2 (COX-2) is overexpressed in cancer. Our objective was to investigate the relationship between COX-2 expression in ovarian carcinoma and clinicopathological factors. An emphasis was put on the association with the new pattern of tumorigenesis that divides tumors into type I--less aggressive, and type II--more aggressive one. The prognostic significance of COX-2 expression was evaluated.METHODS:Ovarian cancer tissues were obtained from 65 patients in FIGO III stage (23 with type I and 42 with type II ovarian cancer). COX-2 expression was evaluated by immunohistochemistry. The statistical analysis was performed in order to assess the connection between COX-2 expression and characteristic factors of ovarian cancer patients as well as the new division for type I and type II ovarian cancerRESULTS:COX-2 expression was detected in 91% of tissue samples. It was markedly elevated in well differentiated tumors (p = 0.0041). The platinum-resistant tumors had significantly higher expression of COX-2 (p = 0.0337). There was no difference between COX-2 expression in type I and type II ovarian cancer (p = 0.6720). The COX-2 staining was not associated to age, CA125 level, the presence of ascites or any special histological type. An increased expression of COX-2 was an unfavorable prognostic factor for overall survival (p = 0.0369) and progression-free survival (p = 0.0218). Multivariate analysis confirmed that COX-2 overexpression is an independent unfavorable prognostic factor of shorter progression-free survival (p = 0.048).CONCLUSIONS:COX-2 expression is an unfavorable prognostic factor for progression-free survival and overall survival in ovarian cancer There is no relationship between COX-2 expression in ovarian cancer tissue and the examined model of ovarian cancer pathogenesis.

SummaryBackgroundInterleukins and NFκ‐B are involved in the development of inflammatory reactions. It has been suggested that these proteins are important contributing factors in the process of photoaging of skin. Moreover, interleukins and NFκ‐B are known to be capable of inducing expression of cyclooxygenase 2 (COX‐2). Expression of COX‐2 in various populations of skin cells has not been examined in the specific processes of aging.ObjectivesThe study aimed at evaluating COX‐2 expression in skin samples originating from patients with chronologically aged and photoaged skin at various stages of skin aging.MethodsImmunohistochemical analysis of COX‐2 reactivity was conducted on samples originating from 52 women undergoing surgery for reasons other than skin pathology.ResultsOur study demonstrated that COX‐2 expression in keratinocytes and fibroblasts was significantly higher in skin samples affected by photoaging than in samples affected by endogenous aging or obtained from younger individuals.ConclusionsThe results indicate that COX‐2 may be involved in the pathogenesis of the photoaging process. Inhibition of expression or activity of the enzyme may find application in photoaging treatment and/or prophylaxis.

Electroporation (EP) can effectively support the penetration of macromolecules from the extracellular space into cells. Electropores induced by the influence of electromagnetic field generate additional paths of transport for macromolecules. The aim of this study was evaluation of the electroporation effect on doxorubicin transport efficiency to human colon (LoVo and LoVo/DX) and gastric (EPG85-257/P and EPG85-257/RDB) adenocarcinoma cells with overexpression of P-glycoprotein and murine macrophage cell line (P388/D1). In our EP experiments cells were placed into a cuvette with aluminum electrodes and pulsed with five square electric pulses of 1300 V/cm and duration of 50 μs each. Cells were also treated with low doxorubicin concentration ([DOX] = 1.7 μM). The ultrastructure (TEM) and changes of P-glycoprotein expression of tumor cells subjected to electric field were monitored. The mitochondrial cell function and trypan blue staining were evaluated after 24 h. Our results indicate the most pronounced effect of EP with DOX and disturbed ultrastructure in resistant gastric and colon cells with decrease of P-gp expression. Electroporation may be an attractive delivery method of cytostatic drugs in chemotherapy, enabling reduction of drug dose, exposure time and side effects.

Epithelial ovarian cancer (EOC) is the most deadly tumor of the female reproductive system. Despite improvements in understanding the biology of EOC, therapeutic strategies still depend on surgery and combination of taxane and platinum agents. Here, we provide a summary of clinically tested biomarkers potentially useful to predict drug response. Resistance against platinum derivatives can result from lower drug concentrations, alterations in the target molecule and changes in the cellular signal transduction pathways. Taxane resistance can develop due to decreased intracellular drug concentration, alterations in microtubuli structure and changes in the cellular response including ERBB2 (epidermal growth factor receptor 2). A few key genes have been suggested as biomarkers for hormonal therapy. Currently, the only targeted therapy agent approved for ovarian cancer is the VEGF (vascular endothelial growth factor) inhibitor bevacizumab. Response to bevacizumab is correlated with VEGF-A levels and hypertension. The primary problems in identifying reliable biomarkers for EOC are the usage of different clinical endpoints, multivariate analysis for a panel of clinical parameters and the lack of published comprehensive clinical information of patients enrolled in these studies. The future lies in adding targeted agents to the taxane/platinum gold standard and in a more detailed stratification of patients into sub-cohorts enabling a more effective therapy. In conclusion, a large-scale coordinated effort is needed for the robust validation of the numerous biomarker candidates available in EOC therapy.

PARP-1 plays an important role in DNA damage repair and maintaining genome integrity by repairing DNA single-strand breaks (SSBs) by base excision repair (BER). The aim of the present study was to examine the expression of PARP-1 in breast cancer (BC) patients and to assess the relationship between the subcellular localization of this protein and clinicopathological characteristics. The reactivity of PARP-1 was analyzed by immunohistochemistry in a homogeneous group of 83 stage II ductal BC patients with a 15-year follow-up. Immunostaining of PARP-1 was also evaluated in 4 human BC cell lines and resistance prediction profile for 11 anticancer agents was performed using 3 models of drug-resistant cell lines. Nuclear-cytoplasmic expression (NCE) was associated with shorter overall survival, which was not statistically significant during the 10-year follow-up but became statistically significant after 10 years of observation, during the 15-year follow-up (P=0.015). Analysis performed in subgroups of patients with (N+) and without (N-) nodal metastases showed that NCE was associated with poor clinical outcome in N- patients (P=0.017). Multivariate analysis confirmed a significant impact of NCE on unfavorable prognosis in N- early BC. The presence of PARP-1 NCE may be a new potential unfavorable prognostic factor in lymph node- negative early BC.

In the last decade, optimized treatment for non-small cell lung cancer had lead to improved prognosis, but the overall survival is still very short. To further understand the molecular basis of the disease we have to identify biomarkers related to survival. Here we present the development of an online tool suitable for the real-time meta-analysis of published lung cancer microarray datasets to identify biomarkers related to survival. We searched the caBIG, GEO and TCGA repositories to identify samples with published gene expression data and survival information. Univariate and multivariate Cox regression analysis, Kaplan-Meier survival plot with hazard ratio and logrank P value are calculated and plotted in R. The complete analysis tool can be accessed online at: www.kmplot.com/lung. All together 1,715 samples of ten independent datasets were integrated into the system. As a demonstration, we used the tool to validate 21 previously published survival associated biomarkers. Of these, survival was best predicted by CDK1 (p<1E-16), CD24 (p<1E-16) and CADM1 (p = 7E-12) in adenocarcinomas and by CCNE1 (p = 2.3E-09) and VEGF (p = 3.3E-10) in all NSCLC patients. Additional genes significantly correlated to survival include RAD51, CDKN2A, OPN, EZH2, ANXA3, ADAM28 and ERCC1. In summary, we established an integrated database and an online tool capable of uni- and multivariate analysis for in silico validation of new biomarker candidates in non-small cell lung cancer.

S100P - low molecular weight acidic protein has been shown to be involved in processes of proliferation, survival, angiogenesis, multidrug resistance and metastasis in various human malignancies. In breast cancer, S100P expression is associated with immortalization of neoplastic cells and aggressive tumour behaviour, indicating that this protein may have adverse prognostic value. We analyzed nuclear and cytoplasmic expression of S100P in 85 stage II breast cancer patients with a median follow up of 17 years. Immunohistochemical reactions were performed on paraffin sections of primary tumours, using monoclonal antibodies against S100P. We also studied prognostic value of S100P mRNA expression using the KM plotter which assessed the effect of 22,277 genes on survival in 2422 breast cancer patients. Moreover, the relationship was examined between expression of S100P in cells of four breast cancer cell lines and their sensitivity to the 11 most frequently applied cytotoxic drugs. Univariate and multivariate analyses showed that higher expression of nuclear S100P (S100Pn) was typical for cases of a shorter overall survival and disease-free time. KM plotter analysis showed that elevated S100P expression was specific for cases of a relapse-free survival and distant metastases-free survival. No relationship could be documented between expression of S100P and sensitivity of breast cancer cells to cytostatic drugs. We demonstrated that a high S100Pn expression level was associated with poor survival in early stage breast cancer patients. Since preliminary data indicated that expression of S100P was up-regulated by activation of glucocorticoid receptor and several agents manifested potential to activate or inhibit S100P promoter activity, this protein might become a therapy target and warrants further studies with respect to its prognostic, predictive and potentially therapeutic value.

In parthanatos, a PARP-1 (poly (ADP-ribose) polymerase 1)-mediated cell death, dissipation of mitochondrial membrane potential, large-scale DNA fragmentation and chromatin condensation were observed. In contrast to apoptosis, it does not cause apoptotic bodies formation. Although PARP-1-mediated cell death presents loss of membrane integrity similar to necrosis, it does not induce cell swelling. The purpose of the study was to correlate the immunohistochemical parameters of PARP-1 reactivity and the selected cytomorphological features of parthanatos: the lack of apoptotic bodies and the absence of necrosis in breast cancer (BC) specimens. Immunohistochemistry for PARP-1 was performed on 83 BC specimens. Correlations between parameters of PARP-1 expression and sub-cellular localisation and the presence of apoptotic bodies and necrosis were evaluated. High expression of PARP-1 (ImmunoReactive Score ≥6) was associated with the lack of apoptotic bodies (P=0.013) and with the absence of necrosis (P=0.002). The presence of apoptotic bodies was correlated with re-distribution of PARP-1 from the nucleus to cytoplasm in BC cells (P=0.029). Additionally, a tendency was observed between necrosis and loss of nuclear PARP-1 expression (P=0.049). Our study suggests that PARP-1 may play a crucial role in induction and regulation of specific type of cellular death called parthanatos.