Sulforaphane-induced apoptosis involves p53 and p38 in melanoma cells.

In malignant melanoma complex reprogramming of cell death and survival pathways leads to increased chemoresistance and poor longer-term survival. Sulforaphane (SF) is a promising isothiocyanate compound occurring in cruciferous plants with reported antiproliferative and proapoptotic activity in several tumor cell lines including melanoma. In this work we investigated the effects of SF in several melanoma cell lines and fresh melanoma cultivates. We found that SF is cytotoxic and induces mitochondrial, caspase-dependent apoptosis in our study model, however with lower efficiency in fresh melanoma cultivates. Moreover, our results indicate that in melanoma cell lines and fresh melanoma cultivates SF induces multiple signaling including oxidative stress-mediated activation of DNA-damage response pathway, changes in p38 kinase activity and enhanced expression of Bax and Puma proapoptotic proteins. In addition, in SF-exposed p53-mutant melanoma cells Puma expression seem to be under p38 control and acts as a compensatory proapoptotic mechanism. Conversely, decreased apoptosis in SF-exposed melanoma cultivates might be attributed to Akt-mediated suppression of p38 as well as p53 activity. Together, our results suggest that SF inhibits growth and proliferation and induces mitochondrial apoptosis both in melanoma cell lines as well as in fresh melanoma cultivates. This proapoptotic effect might be enhanced in combination with Akt inhibitors, in particular in melanoma samples. SF is thus commendable for further preclinical testing, both as a single agent as well as in combination regimens.

Prognostic relevance of angiopoietin-2, fibroblast growth factor-2 and endoglin mRNA expressions in chronic lymphocytic leukemia.

Elevated levels of circulating angiogenic cytokines and increased expression of genes encoding angiogenic factors have been reported in recent years in patients with chronic lymphocytic leukemia (CLL) but data regarding prognostic and predictive significance are still limited. Therefore, in the present study based upon our prior pilot results, we measured mRNA expressions of angiopoietin-2 (Ang-2), fibroblast growth factor-2 (FGF-2) and endoglin (CD105) by reverse transcription quantitative PCR in purified CD19+ cells from 70 untreated CLL patients (median age, 63 years; males, 64%; Rai III/IV stages, 29 %; unmutated IgVH genes, 60 %) and evaluated their possible association with established prognostic factors and clinical course of the disease. Higher expression of Ang-2 was significantly associated with unmutated IgVH genes (n = 55, p = 0.003). Higher CD105 expression was significantly associated with unmutated IgVH genes (n = 55, p < 0.001), high CD38 expression (n = 66, p = 0.022), high ZAP-70 expression (n = 66, p = 0.010), Rai stage I-IV (n = 70, p < 0.001), progressive clinical course of CLL (n = 70, p = 0.001) and shorter time to treatment (n = 70; p < 0.001). Expression of FGF-2 was not significantly associated with any of the prognostic markers. These results indicate that elevated expression of Ang-2 and in particular CD105 by CLL cells is associated with unfavorable prognostic features and clinical outcome; thus, both cytokines appear to play an important role in biology and progression of CLL and warrant further investigation.

[Proteins of resistance and drug resistance in ovarian carcinoma patients]. / Proteiny rezistence a chemorezistence u pacientek s karcinomem ovaria.

BACKGROUND: To evaluate the correlation of resistance proteins LRP (Lung Resistance Protein), Pgp (P-glycoprotein), MRP (Multidrug Resistance-Associated Protein), MRP3 a MRP5 with stage, grade and histological type. To asses correlation of these resistance proteins with drug resistance/drug sensitivity in vitro by means of the MTT assay in ovarian cancer patients. To find the clinical outcome of these data. PATIENTS AND METHODS: 64 women with epithelial ovarian cancer who underwent primary surgery in 2006-2010 had specimens stained with immunohistochemistry for LRP, MRP, MRP3, MRP5 and Pgp and MTT assay (MTT-(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide). RESULTS: Patients with late ovarian cancer had a higher Pgp, MRP, MRP3 and MRP5 level compared to ovarian cancer patients with early stage ovarian cancer. No correlation of resistance proteins with grading was found. Patients with high Pgp and MRP expression had significantly shorter progression-free survival. Patients with drug resistance in vitro by means of the MTT assay had higher Pgp and MRP expression. CONCLUSION: P-glycoprotein and MRP may be useful predictor for outcome of primary chemotherapy in patients with ovarian cancer.

Camptothecin induces p53-dependent and -independent apoptogenic signaling in melanoma cells.

Various DNA-targeting agents may initiate p53-dependent as well as p53-independent response and subsequent apoptosis via alternative cellular systems which include for instance p73, caspase-2 or Bcl-2 family proteins. The scope of involvement of individual molecules in this process and the mechanisms governing their potential interplay are still not entirely understood, in particular in highly aggressive cancers such as in malignant melanoma. In this work we investigated the role and involvement of both p53-dependent and -independent mechanisms in selected melanoma cell lines with differing status of p53 using a model DNA topoisomerase I inhibitor camptothecin (CPT). Here we report that CPT induced in Bowes melanoma cells apoptosis which is essentially p53 and mitochondria-dependent but with some involvement of caspase-2 and p73. Conversely, in mutant p53 melanoma cells overall levels of CPT-induced apoptosis are significantly lower, with p73 and caspase-2 signaling playing important roles. In addition, in these cells the expression of micro RNAs family 34 (miR-34) were low compared to wild-type p53 cells. The ectopic expression of wild type p53 than restored apoptotic response of cells to CPT despite the fact that the expression of miR-34 and miR-155 were not influenced. These results suggest that CPT induces multivariate cellular stress responses including activation of DNA-damage response-p53 pathway as well as p53-independent signaling and their mutual crosstalk play the decisive role in the efficient triggering of apoptosis in melanoma cells.

[Primary drug resistance/sensitivity in vitro and clininical outcome in ovarian cancer patients]. / Primární rezistence/senzitivita in vitro a klinický prubeh onemocnení u pacientek s karcinomem ovaria.

OBJECTIVE: To assay correlation between primary resistance/sensitivity in vitro by MTT test in solid tumor and ascitic fluid and clininical outcome in ovarian cancer patients. DESIGN: Prospective study. SETTING: Department of Gynecology and Obstetrics, Medical Faculty Charles University, Prague and University Hospital, Hradec Králové. METHODS: MTT - (3-(4,5 - Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) chemosensitivity assay was performed in 45 samples of ovarian cancer tissue and 26 samples of ascitic fluid in ovarian cancer patients. We studied the in vitro drug resistance profiles of ovarian cancer specimens exposed to cisplatin, carboplatin, paclitaxel, topotecan, gemcitabin, etoposid. RESULTS: The highest incidence of primary drug resistance in vitro had gemcitabin and carboplatin and the lowest incidence of primary drug resistance had cisplatin and topotecan. Cisplatin had lower incidence of primary drug resistance in vitro than carboplatin. Grade and stage of epithelial ovarian cancer did not correlate to the primary drug resistance/sensitivity in vitro in ovarian cancer patients. The histological subtype of epithelial ovarian cancer correlated to the resistance and sensitivity to chemotherapeutic agents in vitro. Ovarian cancer patients with primary drug resistance to paclitaxel and carboplatin in vitro had more complications during primary chemotherapy, shorter progression free interval and worse prognosis of the disease. CONCLUSION: The lowest incidence of primary drug resistance in vitro had cisplatin. Ovarian cancer patients with in vitro resistance to paclitaxel and carboplatin had significantly higher risk for progression of disease when treated with standard platinum-paclitaxel based regimens. The primary resistance/sensitivity assay would contribute to the targeted treatment and better prognosis of ovarian cancer patients.

Dual inhibition of topoisomerases enhances apoptosis in melanoma cells.

The cytotoxicity of topoisomerase I inhibiting camptothecin, topoisomerase II inhibiting etoposide and their combination were investigated in wild type p53 Bowes and mutant p53 SK-MEL-28 melanoma cell lines during 24h. A combination of camptothecin and etoposide (1 microg/ml + 10 microg/ml) proved to be efficient in both types of cell lines, although mutant p53 cells exhibited a higher resistance. Further studies proved that in Bowes cells, a combination of drugs induced p53-dependent mitochondrial apoptosis characterized by activation of caspases-8 and -2, -9 and -3 with some concurrent involvement of oxidative stress. In SK-MEL-28 cells, apoptosis was found to be mediated via increased oxidative stress, activation of stress kinases such as p38 and SAPK/JNK and mitochondrial dysfunction without significant involvement of p53 and its transactivated target genes. These results demonstrate efficiency of dual inhibition of topoisomerases in melanoma cells with functional as well as mutant p53 and point out at possible further investigation of this modality in preclinical and clinical oncology.

[Resistance/sensitivity in vitro in ovarian cancer patients]. / Rezistence/senzitivita in vitro u pacientek s karcinomem ovaria.

OBJECTIVE: To assay resistance/sensitivity by MTT test in solid tumor or ascitic fluid in ovarian cancer patients. DESIGN: Prospective study. SETTING: Department of Gynecology and Obstetrics, Medical Faculty Charles University, Prague and University Hospital, Hradec Králové. METHODS: MTT - (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) chemosensitivity assay was performed in 32 samples of ovarian cancer tissue and 26 samples of ascitic fluid in ovarian cancer patients. We studied the in vitro drug resistance profiles of ovarian cancer specimens exposed to cisplatin, carboplatin, paclitaxel, topotecan, gemcitabin, etoposid. RESULTS: The highest frequency of resistance in vitro occured for etoposid, gemcitabin and paclitaxel and the most effective chemotherapeutical agents in vitro were cisplatinum and topotecan. Cisplatin had the lowest incidence of drug resistance in vitro than carboplatin. CONCLUSION: Resistance/sensitivity assay would improve the treatment and prognosis of ovarian cancer patients.

[Chemoresistance/Chemosensitivity of ovarian cancer--a case report]. / Chemorezistence/chemosenzitivita ovariálního karcinomu--kazuistika.

OBJECTIVE: To describe chemoresistance and chemosensitivity of two ovarian cancer patients and influence on their medical outcome. SETTINGS: Department of Obstetrics and Gynecology, Medical Faculty Charles University Prague and University Hospital, Hradec Králové. SUBJECT AND METHOD: A case report of two ovarian cancer patients with defined chemoresistance/chemosensitivity of their tumors. We used WST-MTT-1 test for the assessment of chemoresistance/chemosensitivity. CONCLUSION: The chemoresistance/chemosensitivity assay would improve the treatment and prognosis of ovarian cancer patients.

Topoisomerases and tubulin inhibitors: a promising combination for cancer treatment.

Modern approaches to treatment of cancer seek to activate the internal suicide program in the malignant cells, and thereby effectively eliminate them without engaging most of other bodily systems. Many currently used cytostatics are known to induce apoptosis and efforts are being paid to develop new ones with better and more effective proapoptotic potential. Nevertheless, despite recent developments in this field, there are still numerous malignancies showing a varying degree of resistance to cell death due to the corrupted signaling pathways and genetic alterations, often in conjunction with expansive proliferation rate. It has been shown that topoisomerase inhibiting agents such as etoposide, camptothecin and others represent a powerful and dynamic group of cytostatic chemicals used in experimental and clinical conditions. So, it is a group of microtubule targeting poisons comprising classical colchicines on the one hand and new taxanes on the other hand. Since several members of both groups have been evidenced as apoptosis inducers operating via distinct mechanism, their combination should theoretically enhance the final therapeutic outcome. This minireview focuses on the possibilities of such a combinational approach with respect to possible benefits and hazards of this strategy.

Apoptosis - when the cells begin to dance.

We present a 24-hour time-lapse videosequence of in vitro behavior of Hep-2 cells treated with 10 m g/ml Etoposide, a topoisomerase II inhibitor. The cell behavior was recorded by a Mitsubishi video recorder, HS-S5600. In the presented sequence, we show the typical cell rounding accompanied by formation of numerous pseudopodia and rapid rhythmical contractions, so called membrane blebbing known as "dance of death".

3D-computer based reconstructions of apoptotic nuclei.

We present a 3D-model of apoptotic nuclei of HL-60 cells treated with 10 g/ml Etoposide (topoisomerase II inhibitor) for 24 hours. The static model was generated from a series of optical sections obtained through a confocal microscope by freeware and shareware graphical programs available in the Internet. Its animation was done by 3D Studio Max. We demonstrate the appearance of typical fragmentation and condensation of chromatin accompanied by its aggregation to the inner side of the nuclear membrane.

Hydrogels in endovascular embolization. VI. Toxicity tests of poly(2-hydroxyethyl methacrylate) particles on cell cultures.

Cytotoxicity of poly(2-hydroxyethyl methacrylate) [poly(HEMA)] hydrogel spherical particles, prepared by radical suspension polymerization and designed for endovascular occlusion, was studied in vitro on cell cultures. Testing methods included a direct contact test and extraction test. No inhibition of growth of cells surrounding the poly(HEMA) beads and a very low inhibition of cell viability, only in concentrated extracts in long-term contact, were observed. As a result, poly(HEMA) beads can be considered non-toxic.

Radiopaque poly(2-hydroxyethyl methacrylate) particles containing silver iodide complexes tested on cell culture.

Silver iodide complexes have been used as an effective radiopacifying agent to prepare radiopaque poly(2-hydroxyethyl methacrylate) (poly(HEMA)) particles. Incorporation of silver iodide complexes inside the poly(HEMA) particles was achieved by first swelling particles in potassium iodide solution and precipitating the silver iodide complexes using a 30 wt% solution of silver nitrate. The dry particles contained 15 wt% of silver iodide complexes. Particles were easily monitored using a standard imaging technique based on X-ray absorption. Toxicity of the particles has been determined in in vitro experiments on a cell culture. As no inhibition of growth of cells surrounding the particles was observed, they can be considered non-toxic.

Sodium salicylate inhibits NF-kappaB and induces apoptosis in PC12 cells.

Sodium salicylate (NaSal) is an effective analgetic and antiinflammatory drug. Beside its well-known inhibitory effect on the cyclooxigenase enzymes, it influences the activity of other signal transduction proteins including nuclear factor kappa B (NF-kappaB) transcription factor. NF-kappaB is found in the cytoplasm bound to an inhibitory protein, inhibitory kappa B (IkappaB). After its phosphorylation, IkappaB is degraded and the released NF-kappaB translocates into the nucleus. Sodium salicylate inhibits the degradation of IkappaB, thus, NF-kappaB activation cannot occur. According to previous observations, the inhibition of this activation can lead to apoptosis. The main goals of this study were to demonstrate that inhibition of NF-kappaB by sodium salicylate decreases the viability of rat phaeochromocytoma PC12 cells and to investigate the nature of cell damage and death. PC12 cells were treated with different concentrations of sodium salicylate (1-20 mM). Higher concentrations (10-20 mM) killed PC12 cells in a dose-dependent manner. The assessments were done by direct cell counting in a Burker chamber and by the WST-1 cytotoxicity assay. We also found a decreased NF-kappaB activity after sodium salicylate treatment by electrophoretic mobility shift assay (EMSA). The cells treated with sodium salicylate were undergoing apoptosis as seen on our records obtained by time-lapse videomicroscopy as well as shown by DNA fragmentation experiments. The decreased DNA binding activity of NF-kappaB indicates that the inhibition of NF-kappaB can play a role in these processes.

In vitro toxicity testing of implantation materials used in medicine: Effects on cell morphology, cell proliferation and DNA synthesis.

Modern medicine depends in many cases on implantation of xenobiotic materials into the human body. Toxicity evaluation of these materials (plastic, metal alloy, ceramics) is rather difficult, mainly because of their intricate chemical nature and insolubility. There are two main approaches to the toxicity testing of implantation materials: cells are either placed in direct contact with materials to be tested or are treated with eluates made from the materials under study. Cell proliferation and cell morphology were chosen as principal endpoints in general cytotoxicity testing. Two modified methods were used as a measure of cell proliferation: first, direct cell counting within defined areas during subsequent time intervals; secondly, determination of DNA synthesis based on incorporation of radiolabelled thymidine. Prelabelling with [(14)C]thymidine was used and [(3)H]thymidine was applied after treatment with the substance tested. Changes in the (3)H:(14)C ratio are proportional to changes in DNA synthesis. The potential usefulness of all three methods was evaluated by testing a new bioactive ceramic (BAS) to be used in orthopaedics, and a new polymer, EVICROL ESTHETIC, to be used in dentistry. Similar results were obtained with all three methods.

Apoptosis and necrosis: Dynamics of structural changes in cells cultivated in vitro after treatment with xenobiotics.

Cell death has recently become a very widely discussed topic, and a great deal of information has become available regarding physiological (programmed) cell death or apoptosis. Contrary to the situation with apoptosis, few new developments have occurred in the field of xenobiotic-induced cell death. The purpose of this study was to analyse morphological changes related to in vitro induced cell death. The main emphasis was on detailed analysis of the dynamics of structural changes. The experimental models of cell death include both apoptosis (Cisplatin) and necrosis (Tween 20). Cells of stabilized cell line Hep2 (human epithelial cells) were used in this study. The dynamics of structural changes were followed using time-lapse microcinematography of living cells observed in phase contrast. The experiments followed the same population of cells before, during and after treatment with model compounds. It is possible to distinguish characteristic types of cell death in vitro (from morphological criteria), but many intermediate types of cell death can also be found. Cellular responses to the presence of one particular xenobiotic appear to be complex, dependent on the dosage: even in a single treated cell population, different morphological responses were observed.

In vitro cytotoxicity testing of metal alloys used in medicine: Comparison of different approaches.

Metal alloys are used in medicine mainly for implantation in different branches of surgery and as indispensable materials in stomatology. The toxicity testing of these materials incurs even more technical problems than the toxicity testing of new drugs. In principle there are two approaches-direct contact of a tested material with cells, or toxicity testing of an eluate prepared from tested material. We have tested more than 20 different metal alloys, based either on titanium, gold or stainless-steel. We used three different in vitro methods: the dynamic assessment of contact cytotoxicity-cell monolayer is in direct contact with tested material (Cervinka and Puza, 1990); cell proliferation assessment-effect of an eluate on cell proliferation is measured (Cervinka and Puza, 1990); agar overlayer with neutral red uptake-both effect of eluate and direct contact with tested material with agar overlayer could be measured. Based on our results we recommended to use all three approaches simultaneously in a battery of in vitro tests.

Cinematographic study of the effects of cis-diammine-dichloroplatinum on mouse L cells.

Cells of mouse fibroblasts line L were grown one hour in the presence of cisplatin. Their fate was then followed by time-lapse microcinematography during six days. Up to the concentration 3.2 mumol 1(-1) there was little effect observed. Higher concentrations induced prolongation of mitosis and interphase, finally resulting in non-cycling cells with complete and persistent division arrest at 25 mumol 1(-1). After 72 hours following the treatment the cellular and nuclear volumes in non-cycling cells began to grow progressively. The mobility and the ruffling activity of the cell membrane were unaltered. Thus, energy generation, the function of the cytoskeleton, and synthetic processes seemed unchanged. The effect of cisplatin is cytostatic rather than cytotoxic.

Combined effect of sodium selenite and campthotecin on cervical carcinoma cells.

The effects of selenite, campthotecin and their combination were investigated in cervical carcinoma cell line Hep-2 HeLa during 24h. The measured parameters included morphological changes, proliferation, oxidative stress, mitochondrial status, caspase-3 activation and nuclear fragmentation. Selenite at all but lowest concentrations inhibited cell growth and proliferation and induced cell death characterized by membrane blebbing, oxidative stress and mitochondrial damage, occurring in the absence of caspase-3 activation and nuclear fragmentation. Campthotecin at all concentrations induced gradual apoptosis including all measured morphological and molecular parameters with exception of oxidative stress. A combination of selenite and campthotecin induced both antagonistic and synergistic effects on cervical carcinoma cells. While low selenium concentration slightly reduced cytotoxicity and proapoptotic effects of campthotecin, moderate and higher concentrations of selenium enhanced them, changing simultaneously apoptosis into more necrosis-like death. These results show importance of selenium as a potential modulator and enhancer of campthotecin-based anticancer therapy in nonovarian malignancies.

Fusion of L line daughter cells in the presence of Sendai virus.

Sendai virus induces the fusion of daughter cells in a population of murine fibroblasts (L cell line). Mitosis and the ensuing fusion of two daughter cells has been observed by means of time-lapse cinemicrography. The paper documents two types of cell fusion: a) two daughter cells become completely separated at the end of the mitosis, and fuse only after a certain period of time, or b) the daughter cells remain attached through a narrow bridge of cytoplasm which persists between the cells as a consequence of incomplete cytokinesis. Widening of the cytoplasmic bridge ultimately results in the fusion of cells. The interval between the termination of mitosis and the fusion ranged from 45 to 60 min. The biological significance of the resulting polyploid cells is discussed.