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.

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.

[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.

[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.

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.

[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 testing of human ovarian cancer cells and its in vitro model.

Ovarian carcinoma represents the most common cause of death from gynecological malignancies in Europe and North America, being the third most frequent and the first as to the mortality. The standard chemotherapeutical regimen for ovarian cancer involves the administration of platinum derivate (carboplatin, cisplatin), in advanced stage is platinum derivate combined with paclitaxel. Introducing chemoresistance testing of ovarian tumour cells may help to choose optimal chemotherapeutic drug and customize the individual chemotherapeutical regimens in patients. One of approaches of individualization of chemotherapy is in vitro chemosensitivity testing. In our study, we evaluated the cytotoxic effects of selected chemotherapeutics in cells isolated from ovarian tumours and ascites of individual patients. Panel of chemotherapeutics used in the study included cisplatin, paclitaxel, carboplatin, topotecan, gemcitabine and etoposide and their effects on cell viability were determined by the MTT assay. In the total number of 32 clinical samples of tumour and ascites cells, the highest sensitivity showed cells to topotecan, sensitivity to cisplatin was higher than to carboplatin and paclitaxel used in clinical practice showed most often only the marginal reactivity. Resistance to carboplatin and most of the time to gemcitabine and etoposide was commonly present. When the same test on cells that have been frozen for several weeks was repeated it was found that in 20 cases chemosensitivity increased while in 18 cases decreased. In remaining cases there was no change in reactivity to cytostatics. Moreover, chemosensitivity of cells isolated from solid tumour and ascites from the same patient did not show any significant difference with exaption of paclitaxel.

Angiopoietin-2 mRNA expression is increased in chronic lymphocytic leukemia patients with poor prognostic features.

Several studies have demonstrated the potential prognostic importance of angiogenesis in chronic lymphocytic leukemia (CLL). Elevated expression of angiopoietin-2 (Ang-2), an angiogenic cytokine, was recently reported in CLL. However, data regarding prognostic significance of Ang-2 in CLL are limited. Therefore, we quantitated Ang-2 mRNA in purified mononuclear cells of 33 untreated CLL patients and compared the transcript levels to traditional as well as modern prognostic factors in patients with CLL (clinical stage, disease course, IgVH mutation status, CD38, and ZAP-70 expression). Elevated Ang-2 mRNA concentrations were detected in 12 cases; 21 patients had very low or undetectable levels of Ang-2 transcript. There was significant association between high Ang-2 mRNA levels and unmutated IgVH genes (n=27, P=0.010) and with CD38 expression (n=32, P=0.011), but not with ZAP-70 expression (n=32, P=0.784), Rai stage (n=33, P=0.305) or stable versus progressive clinical course (n=33, P=0.443). There was a trend towards shorter progression-free survival in patients with high Ang-2 expression; however, it did not reach statistical significance (P=0.090). Our pilot data show that Ang-2 mRNA is differentially expressed in patients with CLL and its increased expression appears to be associated with poor prognostic features. Further studies are needed to confirm the results in a larger patient cohort.

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.

Effect of phytic acid and inositol on the proliferation and apoptosis of cells derived from colorectal carcinoma.

We characterized the effect of phytic acid (inositol hexaphosphate, IP6) as a potential adjuvant in treatment of colorectal carcinoma and evaluated the optimal concentration and treatment time to produe the maximal therapeutic effect. There is some evidence that myoinositol (Ins) can potentiate anti-cancer effects of IP6. Therefore, we tested both IP6 and Ins individually and in combination on human cell lines HT-29, SW-480 and SW-620 derived from colorectal carcinoma in different stages of malignancy. The effect of tested chemicals on the cells was measured using metabolic activity assay (WST-1), DNA synthesis assay (BrdU), protein synthesis assay (Brilliant Blue) and apoptosis (caspase-3 activity). We tested IP6 and Ins at three concentrations: 0.2, 1 and 5 mM for 24, 48 and 72 h. The concentrations and incubation periods were chosen according to low toxicity of the tested substance that was observed in a long-term clinical study. We found that all employed concentrations of IP6 or IP6/Ins decreased proliferation of the cell lines, with the maximum decrease being observed in HT-29 cells. Metabolic activity of treated cells differed in response to IP6 and IP6/Ins treatment; in HT-29 and SW-620 significant decrease was observed only at the highest concentration, whereas in SW-480 cells metabolic activity was lower at each concentration except 0.2 and 1 mM IP6 or IP6/Ins in 24-h incubation. The results from protein content assay corresponded to the results obtained from WST assay. In addition, we found maximum increase in caspase-3 activity at concentration 5 mM IP6 or IP6/Ins in HT-29 cells and with IP6 at concentration of 0.2 mM or IP6/Ins in SW-480 cells with clear indication of Ins enhancing the proapoptotic effect of IP6 in all the cell lines studied.

Activation of several concurrent proapoptic pathways by sulforaphane in human colon cancer cells SW620.

Despite the reported cytotoxicity and apoptosis-inducing properties of sulforaphane (SF) in colon cancer cells, the details concerning individual mechanisms and signaling cascades underlying SF-mediated apoptosis remain unclear. To understand different aspects of SF-induced proapoptic signaling in advanced colon carcinoma, we investigated its mechanisms in metastatic SW620 cell line. Our results indicate that in SW620 cells SF acts to induce multivariate cascades including DNA-damage response pathway whose proapoptotic signaling is nevertheless reduced owing to the mutant status of p53 and caspase-2-JNK pathway which seems to complement and enhance p53-dependent signaling, however only in wild-type p53. Furthermore, both pathways require the active role of mitochondria and do not depend on generation of ROS, making SF an attractive chemopreventive agent whose antitumor properties should be further investigated in colon cancer.

[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.

Selenium activates p53 and p38 pathways and induces caspase-independent cell death in cervical cancer cells.

The mechanisms of sodium selenite-induced cell death in cervical carcinoma cells were studied during 24 h of exposure in the HeLa Hep-2 cell line. Selenite at the employed concentrations of 5 and 50 micromol/L produced time- and dose-dependent suppression of DNA synthesis and induced DNA damage which resulted in phosphorylation of histone H2A.X. These effects were influenced by pretreatment of cells with the SOD/catalase mimetic MnTMPyP or glutathione-depleting buthionine sulfoximine, suggesting the significant role of selenite-generated oxidative stress. Following the DNA damage, selenite activated p53-dependent pathway as evidenced by the appearance of phosphorylated p53 and accumulation of p21 in the treated cells. Concomitantly, selenite activated p38 pathway but its effect on JNK was very weak. p53- and p38-dependent signaling led to the accumulation of Bax protein, which was preventable by specific inhibitors of p38 (SB 203580) and p53 (Pifithrin-alpha). Mitochondria in selenite-treated cells changed their dynamics (shape and localization) and released AIF and Smac/Diablo, which initiated caspase-independent apoptosis as confirmed by the caspase-3 activity assay and the low effect of caspase inhibitors z-DEVD-fmk and z-VAD-fmk on cell death. We conclude that selenite induces caspase-independent apoptosis in cervical carcinoma cells mostly by oxidative stress-mediated activation of p53 and p38 pathways, but other selenite-mediated effects, in particular mitochondria-specific ones, are also involved.

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.

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.

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.

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.

Toxic effects of chromium acetate hydroxide on cells cultivated in vitro.

Many human activities, particularly industrial ones, result in an ever-growing production of toxic waste materials. The dynamics of the toxic effects of chromium acetate hydroxide, which is found in high concentrations in a waste sediment produced in the Czech Republic, were assessed by using a battery of in vitro tests carried out on two cell lines: L-929 (mouse fibroblasts) and Hep 2 (human laryngeal cells). Various markers of cell damage were assessed by phase-contrast, video and fluorescence microscopy, fluorometry, and DNA analysis. Chromium acetate hydroxide, over a concentration range of 1-0.02mol/l induced immediate cell death by fixation, whereas, at 0.002mol/l, the treated cells died in a much slower, more discrete manner. All the detected markers of cell damage, whether immediate or slow, clearly demonstrated that the cells died by necrosis. On the other hand, test concentration of 0.001mol/l appeared to constitute a threshold at which no pathological changes of Hep 2 cells were observed over 96 hours. We conclude that chromium acetate hydroxide has a high toxic potential in vitro, which should be considered when studying the toxicity of waste materials containing it.

[Zinc and its role in the regulation of cell death]. / Zinek a jeho role pri regulaci bunecné smrti.

Zinc is a key element for maintenance of the structural and functional integrity of eukaryotic cells and tissues. In living systems, it forms stable complexes with macromolecules as well as so called labile pools called zincosomes, which are nowadays considered crucial for the regulation of apoptosis and cell proliferation. Zinc may block apoptosis induced by many external factors by inhibiting caspases and endonucleases, through interactions with transcription factors and kinases or due to its antioxidant activities. On the other hand, depletion of zinc may lead to rapid activation of apoptotic cascade and consequent cell death in many types of cells. Imbalances in intracellular zinc pools lead to improper regulation of cell death and proliferation, which is often causing or accompanying diseases. Therefore, detailed elucidation of the role of zinc in these regulations presents a solution for various pathophysiological conditions.

The dynamics of the hexavalent chromium induced apoptotic patterns in vitro.

Although hexavalent chromium has been shown to induce apoptosis in cells cultivated in vitro, there appear to be no studies focusing on the dynamics of this process. To find out about dynamic patterns of hexavalent chromium-induced apoptosis, we treated Hep2 cells with 150 micrograms/ml potassium chromate and recorded their behavior as well as appearance of some crucial organelles using different morphological and biochemical methods. We found that Hep2 cells showed the earliest observable changes at 6 hours after the treatment (blebbing, chromatin shrinkage), with the entire apoptotic process lasting up to 24 hours. While all the observed cell features clearly prove apoptosis induced by hexavalent chromium, a typical apoptotic hallmark, DNA ladder, seems not to occur in this type of cells. On the other hand, in HL60 cells, used as a control, this ladder was observable.