Evaluation of transfection effectiveness using fluorescein-labelled oligonucleotides and various liposomes

Silencing of genes using siRNA represents a generally used technique aimed at inhibiting expression of proteins in cells. Results have frequently not met expectations and this has posed problems in association with this technique. The phenomenon might reflect an incorrect sequence of RNAi, poor penetration of the cells by the nucleotides or insufficient knowledge of the protein in question. The present study is aimed at evaluating the effectiveness of the transfection of selected cell lines using various liposomes. The studies were performed using 9 cell lines: EPG 257/85 RNOV, EPG 257/85 RDB, W 181/A17, A 2780P, A 2780 RCIS, MEWO CIS, 181 RDB, 181 P and MCF-7/MX. The lines were transfected with fluorescently labelled oligonucleotides. Two parallel experiments were performed. In one oligofectamin and in the other DMRI were used as oligonucleotide carriers. The studies demonstrated that in every case nucleotides penetrated more than 90% of the cells. In 4 cell lines the signal was stronger when oligofectamin was used, in 4 cell lines when DMRI was employed and in one case the signal strength was comparable using any carrier. The studies showed that various liposomes demonstrated distinct transfection efficiency, depending on the cell line used, and that application of fluorescently labelled nucleotides may be helpful in the optimisation of transfection conditions

Kwercetyna hamuje proliferację i zwiększa wrażliwość komórek raka jajnika na cisplatynę i paklitaksel

Introduction: Due to frequent diagnosis of ovarian cancer at an advanced clinical stage, in most cases surgical debulking is followed by chemotherapy. The principal cause of therapeutic failure involves incomplete surgery and resistance of neoplastic cells to chemotherapy. A search continues for substances which would overcome resistance to treatment and, as a result, would increase efficacy of the applied treatment. Quercetin represents one of more interesting compounds, which at present in subjected to several tests. Material and methods: Studies were performed on in vitro sensitivity of human ovarian cancer cell lines, SKOV-3, EFO27, OVCAR-3 and A2780P to low doses of quercetin and on the effect exerted by quercetin on sensitivity of the cell lines to cisplatin and pactitaxel. Results: The experiments proved that the studied cells of ovarian cancer manifest a similar sensitivity to quercetin. Following incubation of the cells with two distinct concentrations of quercetin and the studied cytostatic agents all the cells lines were found to significantly increase their sensitivity to pactitaxel In cases of two cell lines, OVCAR-2 and A2780P, they also significantly increased their sensitivity to cisplatin. Discussion: Our results demonstrated suitability of low quercetin doses (achievable using oral administration) as a substance which increases sensitivity of ovarian cancer cells to cisplatin and paclitaxel. The value of quercetin include its wide accessibility, efficacy and a broad range of activity but also its low toxicity, as compared to other examined compounds. Conclusions: Used in low doses, quercetin increases chemosensitivity of ovarian cancer cells.Wstęp: Ze względu na częste rozpoznawanie raków jajnika w zaawansowanym stadium klinicznym w większości przypadków po przeprowadzeniu zabiegu operacyjnego stosowana jest chemioterapia. Podstawową przyczyną niepowodzeń stosowanej terapii jest nieradykalność leczenia operacyjnego oraz oporność komórek nowotworowych na chemioterapię. Poszukuje się substancji, które pozwolą zwalczyć oporność na leczenie i w efekcie zwiększyć skuteczność stosowanej terapii. Jednym z ciekawszych związków poddawanych obecnie szeregowi badań jest kwercetyna. Materiał i metody: Przeprowadzono badania in vitro wrażliwości linii komórkowych ludzkiego raka jajnika SKOV-3, EFO27, OVCAR-3 i A2780P na niskie dawki kwercetyny oraz ocenę wpływu kwercetyny na wrażliwość linii komórkowych na cisplatynę i paklitaksel. Wyniki: Przeprowadzone doświadczenia wykazały, że badane komórki raka jajnika wykazują zbliżoną wrażliwość na kwercetynę. W wyniku inkubacji badanych komórek z dwoma różnymi stężeniami kwercetyny i z badanymi cytostatykami wykazaliśmy, że wszystkie linie istotnie zwiększyły swoją wrażliwość na paklitaksel. W przypadku dwóch linii – OVCAR-2 i A2780P uzyskaliśmy również istotny wzrost wrażliwości na cisplatynę. Dyskusja: Nasze badania wykazały przydatność niskich dawek kwercetyny (osiągalnych przy podaży doustnej), jako substancji zwiększającej wrażliwość komórek raka jajnika na cisplatynę i paklitaksel. Jej walory podkreśla nie tylko łatwa dostępność, skuteczność i szeroki zakres działania, ale również mała, w porównaniu z innymi badanymi substancjami, toksyczność. Wnioski: Kwercetyna zastosowana w niskich dawkach powoduje wzrost chemiowrażliwości komórek raka jajnika

The expression of metallothionein (MT) and proliferation intensity in ovarian cancers treated with cisplatin and paclitaxel

Metallothioneins (MT) represent low molecular weight proteins that are supposed to fulfil several functions. They participate in the cell cycle, protect cells from oxidative stress, control levels of heavy metals and participate in multidrug resistance processes, particularly in cases of alkylating drugs. The present study aimed at evaluation of proliferation intensity (Ki67, PCNA) in ovarian cancers treated using cisplatin and paclitaxel, as related to expression of MT. The experiments were performed on samples originating from 10 patients operated on due to ovarian cancer. The material originated from the first operations or second-look operations. All the patients were treated with cisplatin and paclitaxel. Immunocytochemical reactions using antibodies to MT, Ki67 and PCNA were performed in paraffin sections originating from the cases studied. Statistical analysis was performed using Statistica software. The studies demonstrated no relation between expression of MT on the one hand and intensity of proliferation before or after chemotherapy on the other hand (gamma correlation, p > 0.05). The results indicate that expression of MT is not related to resistance to treatment using cisplatin and paclitaxel

Localisation of exogenous surfactants in cell membranes in the air-blood barrier : rat model

The use of exogenous surfactants has been introduced into the therapy of patients of different ages. Much better results have been obtained in the treatment of respiratory distress syndrome with surfactants enriched with surfactant proteins. In the following study we used protein-containing surfactants (survanta and curosurf). The aim of the following study was to determine the localisation of artificial surfactants in the lung tissue. Using the Immunogold Technique, biotinylated surfactant proteins were traced in the air-blood barriers. In all lungs the exogenous surfactant was present only in some alveoli. In these parts small areas of atelectasis as well as oedema and transudate accumulation were seen. These changes were less severe after biotinylated curosurf treatment. In electron microscope studies we found surfactant elements in the air-blood barrier and other structures of the alveolar septa. Immunogold studies confirm the presence of biotynylated surfactant in the elements of the air-blood barrier

Expression of metallothionein (MT) and gluthatione s-transferase pi (SGTP) in the bone marrow of patients with myeloproliferative disorders (MPD)

Overexpression of SGTP and/or MT may contribute to various carcinogenic processes and to resistance to anticancer treatment. The importance of these proteins, although clearly established in solid tumours, has not been fully understood in haematopoietic neoplasm. The aim of this study was to determine the expression of MT and SGTP in the bone marrow of patients with MPD. Twenty paraffin-embedded bone marrow core biopsy specimens from newly diagnosed patients with MPD were evaluated — osteomyelofibrosis (OMF), n = 9 and chronic myelocytic leukaemia (CML), n = 11. We demonstrate increased SGTP and MT expression in the bone marrow of MPD patients. In our study levels of MT in OMF patients were higher than in CML. This suggests that MT expression may correlate with bone marrow fibrosis. These data, although based on a relatively small number of patients, raise the possibility that SGTP and MT may play a role in the pathogenesis of MPD. The clinical significance of this phenomenon needs further investigation

Localisation of exogenous surfactants in cell membranes in the air-blood barrier: rat model

The use of exogenous surfactants has been introduced into the therapy of patients of different ages. Much better results have been obtained in the treatment of respiratory distress syndrome with surfactants enriched with surfactant proteins. In the following study we used protein-containing surfactants (survanta and curosurf). The aim of the following study was to determine the localisation of artificial surfactants in the lung tissue. Using the Immunogold Technique, biotinylated surfactant proteins were traced in the air-blood barriers. In all lungs the exogenous surfactant was present only in some alveoli. In these parts small areas of atelectasis as well as oedema and transudate accumulation were seen. These changes were less severe after biotinylated curosurf treatment. In electron microscope studies we found surfactant elements in the air-blood barrier and other structures of the alveolar septa. Immunogold studies confirm the presence of biotynylated surfactant in the elements of the air-blood barrier

COX-2 expression pattern is related to ovarian cancer differentiation and prognosis, but is not consistent with new model of pathogenesis

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 cancer. Results: 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

Stromal myofibroblasts in breast cancer: relations between their occurrence, tumor grade and expression of some tumour markers.

It is suggested that tumour stromal myofibroblasts exert an unfavourable effect on the biology of breast cancer. We are aware of only a single study which examined relationships between manifestation of myofibroblasts in the stroma of breast cancer and clinicopathological data of the patients. The present study was aimed at estimation of the effect exerted by myofibroblasts present in the tumour stroma on principal pathological parameters and on expression of Ki67, P53 and HER-2 proteins in the group of the most frequent breast cancers, the ductal cancers. In paraffin sections of 60 ductal breast cancers (20 cases in G1, 20 in G2 and 20 in G3), immunohistochemical reactions were performed to detect expression of smooth muscle actin (SMA) in order to visualize myofibroblasts, Ki67, P53 and HER-2. The studies demonstrated that the most numerous myofibroblasts were present in G3 cases and they were the least frequent in G1 cases (P = 0.02). Positive correlations were observed between the presence of myofibroblasts in tumour stroma and expression of Ki67 and HER-2 in breast cancer cells in the entire group (P < 0.001 and P = 0.001, respectively), in G2 cases (P = 0.003 and P = 0.03) and in G3 cases (P = 0.01 and P = 0.03). Considering that the higher grade, Ki67 and HER-2 are thought to represent unfavourable prognostic factors, the elevated content of myofibroblasts in tumour stroma is probably typical for cases with worse prognosis

Analiza ekspresji hMLH1 i hMSH2 u pacjentek z rakiem jajnika leczonych za pomocą pochodnych platyny

Background: Loss of DNA mismatch repair may result in resistance to platinum- based anticancer drugs. The hMLH1 and hMSH2 proteins play a critical role in the maintenance of genome integrity and are involved in resistance to platinum-based therapy in colorectal cancer, which is deficient in hMLH1 protein and endometrial cancer, as well as in hMSH2 protein. However, the predictive value of MLH1 and MSH2 expression in ovarian cancer cisplatinresistance is still to be determined. Objective: The aim of this study was to investigate the expression of hMLH1 and hMSH2 proteins in ovarian carcinoma specimens and to evaluate their prognostic significance by means of overall survival (OS) and progression-free survival rates (PSF). Material: Ovarian cancer tissues were obtained from 61 patients: 45 platinum-sensitive and 16 platinum-resistant. hMLH1 and hMSH2 proteins expression was evaluated by immunohistochemistry, with the use of mouse monoclonal antibodies clone 14 for hMLH1 and clone FE11 for hMSH2. The log-rank test and Kaplan-Meier statistics were used to analyze the relationship between proteins expression and progression free survival, as well as the overall survival. Result: No significant correlation was found between hMLH1 and hMSH2 expression and overall survival and progression free survival in the group of patients sensitive and resistant to cisplatin. No significant difference was found in proteins expression intensity between the two compared groups of patients. Age of patients, type of cancer histology, FIGO staging, grading, clinical response and CA 125 did not reveal correlation with the expression of the analyzed proteins. Conclusion: The immunohistochemical expression of hMLH1 and hMSH2 proteins in ovarian cancer has no predictive value in resistance to cisplatin.Wstęp: Białka hMLH1 i hMSH2 pełnią kluczową rolę w naprawie genomu. Utrata tej zdolności może powodować zwiększoną oporność na leki przeciwnowotworowe oparte na pochodnych platyny. Jakkolwiek wartość prognostyczna oznaczania ekspresji białek hMLH1 i hMSH2 w raku jajnika w celu przewidywania oporności na cisplatynę pozostaje wciąż nieokreślona. Cel pracy: Celem tego opracowania było zbadanie ekspresji białek hMLH1 i hMSH2 w komórkach raka jajnika pochodzących od 61 pacjentek, w tym: 45 wrażliwych i 16 opornych na cisplatynę oraz ocena wpływu tych białek na ogólne przeżycie i czas wolny od wznowy. MateriaΠ i metody: Ekspresję białek hMLH1 i hMSH2 oceniono metoda immunohistochemiczna przy użyciu mysich przeciwciał monoklinalnych, klon 14 dla hMLH1 i klon FE11 dla hMSH2. Do analizy zale˝noEci pomi´dzy ekspresja białek a czasem wolnym od wznowy i przeżyciem ogólnym użyto testu log rank i statystyki Kaplana-Meiera. Wartość

Analiza ekspresji BCRP u pacjentek z rakiem piersi

Breast cancer resistance protein (BCRP, ABCG2) is a xenobiotic half-transporter protein. It is a member of the ATPbinding cassette protein family and functions as an energy-dependent efflux pump. BCRP is involved in multidrug resistance. The study aimed at examining BCRP expression in breast cancers and at defining a relationship between activity of this protein and clinical course of the cancer. Materials and Methods: We analyzed the expression of BCRP in 101 stage II breast cancer patients. All the patients were diagnosed and treated at the Lower Silesia Oncology Centre (LSOC) between January 1993 and June 1994. After the treatment the patients remained under constant control at LSOC. Mean duration of the observation was 14.2 years (ranging between 9.1 and 16.5 years). Data related to relapse of the disease and deaths were obtained from medical documentation stored in LSOC. The immunohistochemical reactions were performed on paraffin sections of primary tumours, using monoclonal antibodies against BCRP. The intensity of immunohistochemical reactions with BCRP antibody was evaluated using the semi-quantitative IRS (ImmunoReactive Score) scale, which took into account the intensity of the colour reaction and percentage of positive cells. Results of the immunohistochemical reactions, pathological and of clinical observations were subjected to statistical analysis. Correlations between these factors and BCRP were analyzed using Spearman and Chi2 tests. In order to estimate the survival rate, we used Kaplan Meier statistics, log-rank tests and Cox proportional hazard regression. Results: In our analysis we observed a positive correlation between the expression of the BCRP protein and grade of tumour advancement (r=0.2 p=0.03). We found also a negative correlation between the expression of BCRP and the estrogen (r=0,24 p=0,02) and progesteron (r=0,28 p=0,02) receptors. In a univariate analysis a significantly shorter disease free survival (DFS) and disease specific survival (DSS) was noted in patients with metastases to the lymph nodes (p=0,003 and p=0,0006), over the age of 50 years old ((p=0,02 and p=0,04) and clearly statistically significant in patients with a high expression of BCRP (p=0,00044 and p=0,00005). Overall survival (OS) was shorter in patients over the age of 50 (p=0,01), with higher stage of the disease – IIB (p=0,025), with metastases to the lymph nodes (p=0,003) and also clearly statistically significant in patients with a high expression of BCRP (p=0.00004). A multivariate analysis allowed to reveal that only higher expression of BCRP and metastases to lymph nodes were typical for cases of DFS (p=0,0028 and p=0,00015), DSS (p=0,00052 and 0,000017) and OS (p=0,0018 and p=0,000007) time. Conclusions: We demonstrated that high BCRP expression level is associated with poor survival in early stage breast cancer patients.Cel pracy: Celem naszej pracy była ocena ekspresji białka BCRP w wycinkach pochodzących od pacjentek chorych na raka piersi i określenie jej związku z odległymi wynikami leczenia. Materiał i metody: Materiał do badań immunohistochemicznych pochodził od 101 pacjentek z rozpoznaniem raka piersi. Badania immunohistochemiczne przeprowadzono na skrawkach parafinowych, z użyciem monoklonalnych przeciwciał przeciwko BCRP. Uzyskane wyniki oraz dane kliniczne pacjentów poddano statystycznej analizie. Wyniki: W jedno i wieloczynnikowej analizie wyników wykazano, że wyższa ekspresja białka BCRP była związana z krótszym czasem przeżycia wolnego od wznowy (DFS), przeżycia specyficznego dla nowotworu (DSS) oraz przeżycia całkowitego (OS). Wnioski: U pacjentek z wczesną postacią raka piersi wysoka ekspresja białka BCRP związana jest z gorszym rokowaniem