First report on parasites of European beavers in the Slovak Republic.

European beaver (Castor fiber L. 1758) is the biggest rodent living in Europe. It is a semi-aquatic animal known for building dams and burrows. European beaver is a potential host for a wide range of parasites and other infectious diseases. In Slovakia, there is an increasing number of beavers but the data about their parasitic fauna are missing. Our work is the first documentation about the beaver's parasitofauna in Slovakia. In a 1-year study, we collected and examined 19 beaver fecal samples from the vicinity of beaver burrows inhabiting three particular localities at the Danube, Topla, and Laborec rivers in Slovakia. In these fecal samples, 4 different species of intestinal endoparasites were detected as follows: oocysts of Cryptosporidium, cysts of Giardia, eggs of Stichorchis subtriquetrus, and eggs and larvae of Travassosius rufus. Parasites were confirmed only in samples collected at river Topla. Based on our results, we can conclude that European beaver can be an important source of parasitic contamination of surface waters especially in the localities shared by people.

Down-regulation of Bcl-2 and Akt induced by combination of photoactivated hypericin and genistein in human breast cancer cells.

Presented experiment considers combination of genistein and photodynamic therapy with hypericin with a view to achieve higher therapeutic outcome in human breast adenocarcinoma cell lines MCF-7 and MDA-MB-231, both identified in our conditions as photodynamic therapy resistant. Since genistein is known to suppress Bcl-2 expression, we predicted that photodynamic therapy with hypericin might benefit from mutual therapeutic combination. In line with our expectations, combined treatment led to down-regulation of Bcl-2 and up-regulation of Bax in both cell lines as well as to suppression of Akt and Erk1/2 phosphorylation induced by photoactivated hypericin in MCF-7 cells. Although Akt and Erk1/2 phosphorylation was not stimulated by photodynamic therapy with hypericin in MDA-MB-231 cells, it was effectively suppressed in combination. Variations in cell death signaling favoring apoptosis were indeed accompanied by cell cycle arrest in G(2)/M-phase, activation of caspase-7, PARP cleavage and increased occurrence of cells with apoptotic morphology of nucleus. All these events corresponded with suppression of proliferation and significantly lowered clonogenic ability of treated cells. In conclusion, our results indicate that pre-treatment with tyrosine kinase inhibitor genistein may significantly improve the effectiveness of photodynamic therapy with hypericin in MCF-7 and MDA-MB-231 breast cancer cells.

Mechanisms involved in the cell cycle and apoptosis of HT-29 cells pre-treated with MK-886 prior to photodynamic therapy with hypericin.

In our previous study we have proved that colon cancer cells HT-29 pre-treated with specific 5-lipoxygenase inhibitor MK-886 became more susceptible to photodynamic therapy (PDT) with hypericin and we also found that this mutual combination induced cell cycle arrest and stimulated onset of apoptosis (Kleban et al., 2007. J. Photochem. Photobiol. B 84, 2). To further explain events associated with MK-886 mediated sensitization of tumor cells toward PDT with hypericin, more detailed study of signaling pathways leading to increase in apoptosis as well as cell cycle perturbations was performed and is presented herein. Intensive accumulation of HT-29 cells in G0/G1 phase of cell cycle led to expression analyses of several G0/G1 checkpoint molecules (cyclin A, cyclin E, cdk-2, pRb). Similarly, accumulation of apoptotic cells invoked analyses of key molecules involved in apoptotic signaling (caspase-3, -8, -9; PARP; Lamin B; Mcl-1; Bax) by Western blotting and caspase activity assay. Long term survival of cells was examined by clonogenicity test. As the effect of PDT is mediated by ROS production, levels of hydrogen peroxides and superoxide anion were monitored by flow cytometric analyses. In addition, an impact of MK-886 on LTB4 production and expression of 5-LOX was monitored. Massive G0/G1 arrest in the cell cycle accompanied by increase in cyclin E level and decrease/absention of cyclin A, cdk-2 and pRb expression indicated incapability for G1/S transition. Minimal changes in cleavage of procaspases observed in cells treated with non-toxic concentrations of either agent alone or their mutual combination were not quite in line with their activity (caspase-3, -8, -9) which was significantly increased mainly in combinations. Treatment with non-toxic concentration of MK-886 had minimal influence over ROS production compared to control cells. In contrast, hypericin alone markedly increased the level of ROS, but no additional effect of MK-886 pre-treatment was detected. Further analyses of particular ROS groups unveiled an impact of increasing MK-886 concentration on superoxide accumulation accompanied with depletion of hydrogen peroxide level within the cells. The clonogenicity test revealed disruption of colony formation after mutual combination of both agents as compared to MK-886 or PDT alone. In conclusion, we presume that stimulation of apoptosis in our experimental model was accomplished preferentially through the mitochondrial pathway, although caspase-8 activation was also noticed. Interestingly, pre-treatment with MK-886 modulated distribution of ROS production in mutual combination with PDT.

Erythropoietin inhibits apoptosis induced by photodynamic therapy in ovarian cancer cells.

Recombinant human erythropoietin is widely used to treat anemia associated with cancer and with the myelosuppressive effects of chemotherapy, particularly platinum-based regimens. Erythropoietin is the principal regulator of erythroid cell proliferation, differentiation, and apoptosis. Recently, the antiapoptotic and proliferative effects of erythropoietin on nonhematopoietic cells were also established. We now show the effect of erythropoietin treatment on the response of A2780 and SKOV3 ovarian carcinoma cell lines to photodynamic therapy (PDT) using hypericin. SKOV3 exhibited an increased resistance to hypericin when cells were treated with erythropoietin. This resistance was reversed by treatment of SKOV3 cells with the specific Janus kinase 2 kinase inhibitor AG490 or the tyrosine kinase inhibitor genistein. These results support a role for the specific erythropoietin-induced Janus kinase 2/STAT signal transduction pathway in PDT resistance. Evidence of erythropoietin signaling was obtained by the demonstration of Akt phosphorylation in both A2780 and SKOV3 cells. Erythropoietin-treated SKOV3 cells exhibited decreased apoptosis induced by hypericin, an effect that was blocked by the phosphoinositide 3-kinase/Akt inhibitor wortmannin. These results may have important implications for ovarian cancer patients undergoing PDT and receiving erythropoietin.

Modulation of hypericin photodynamic therapy by pretreatment with 12 various inhibitors of arachidonic acid metabolism in colon adenocarcinoma HT-29 cells.

One proposal to increase the efficiency of photodynamic therapy (PDT) is to accompany photosensitization with other treatment modalities, including modulation of arachidonic acid (AA) metabolism. The aim of this study was to evaluate the effectiveness of a combined modality approach employing 48 and 24 h pretreatment with various inhibitors of lipoxygenase (LOX; nordihydroguaiaretic acid, esculetin, AA-861, MK-886 and baicalein), cyclooxygenase (COX; diclofenac, flurbiprofen, ibuprofen, indomethacin, SC-560 and rofecoxib) and cytochrome P450-monooxygenase (proadifen) pathways, followed by hypericin-mediated PDT. Cytokinetic parameters like MTT assay, adherent and floating cell numbers, viability and cell cycle distribution analysis were examined 24 h after hypericin activation. Pretreatment of human colon cancer cells HT-29 prior to PDT with 5-LOX inhibitor MK-886 as well as 5, 12-LOX and 12-LOX inhibitors (esculetin and baicalein, respectively) resulted in significant and dose-dependent effects on all parameters tested. Pretreatment with diclofenac, flurbiprofen, ibuprofen and indomethacin, the nonspecific COX inhibitors, promoted hypericin-mediated PDT, but these effects were probably COX-independent. In contrast, application of SC-560 and rofecoxib, specific inhibitors of COX-1 and COX-2, respectively, attenuated PDT. Inhibition of P450 monooxygenase with proadifen implied also the significance of this metabolic pathway in cell survival and cell resistance to hypericin photocytotoxicity. In conclusion, our results testify that application of diverse inhibitors of AA metabolism may have different consequences on cellular response to hypericin-mediated PDT and that some of them could be considered for potentiation of PDT.

Necrosis predominates in the cell death of human colon adenocarcinoma HT-29 cells treated under variable conditions of photodynamic therapy with hypericin.

Photodynamic therapy (PDT) represents a new rapidly-developing anticancer approach based on administration of a non- or weakly-toxic photosensitizer and its activation with light of appropriate wavelength. Hypericin, one of the promising photosensitizers, is known to induce apoptosis with high efficiency in various cell line models. However, here we report the prevalence of necrosis accompanied by suppression of caspase-3 activation in colon adenocarcinoma HT-29 cells exposed to an extensive range of PDT doses evoked by variations in two variables -- hypericin concentration and light dose. Necrosis was the principal mode of cell death despite different PDT doses and the absence of anti-apoptotic Bcl-2 expression, even if the same condition induced caspase-3 activity at similar toxicity in HeLa cells. Introduction of Bcl-2 into HT-29 cells invoked caspase-3 activation, changed the Bcl-X(L) expression pattern, increased the apoptosis ratio with no effect on overall toxicity, and supported arrest in the G(2)/M-phase of cell cycle. Since it is known that Bcl-2 suppression in HT-29 is reversible and linked to the over-expression of mutated p53 and also considering our data, we suggest that the mutation in p53 and events linked to this feature may play a role in cell death signalling in HT-29 colon cancer cells.

Hypericin-induced photocytotoxicity is connected with G2/M arrest in HT-29 and S-phase arrest in U937 cells.

Susceptibility of the HT-29 human colon adenocarcinoma cell line and human myeloid leukemia cell line U937 to hypericin-mediated photocytotoxicity was investigated and compared in this study. Cellular parameters as viability, cell number, metabolic activity and total protein amount were monitored in screening experiments with subsequent cell-cycle analysis and apoptosis detection to determine the cellular response of the different tumor types to various concentrations of photoactivated hypericin. The results show concentration dependence of the photosensitizer's cytotoxicity on the studied cell lines, with higher sensitivity of U937 cells. Whereas the two extreme hypericin concentrations (1 x 10(-9) M and 1 x 10(-6) M) resulted in similar changes in all tested cellular parameters on the two studied cell lines, 1 x 10(-8) M and 1 x 10(-7) M hypericin treatment resulted in different responses of the cell lines in all monitored parameters except for viability. Although leukemic cells proved sensitive to both 1 x 10(-8) M and 1 x 10(-7) M hypericin, significant changes on HT-29 cells were detected only after the 1 x 10(-7) M hypericin concentration. Cell-cycle arrest was related to simultaneously occurring apoptosis in colon cancer. Remarkable is the difference in cell-cycle profile where G2/M arrest in colon cancer cells versus accumulation of leukemic cells in the S phase appears. This suggests that hypericin treatment affecting the cell-cycle machinery of different cancer cells is not universal in effect.

Pre-treatment of HT-29 cells with 5-LOX inhibitor (MK-886) induces changes in cell cycle and increases apoptosis after photodynamic therapy with hypericin.

It may be hypothesized that the lipoxygenase (LOX) metabolic pathway plays an important role in photodynamic therapy (PDT) of malignant tumours, and modification of this pathway may result in administration of lower doses of photodynamic active agents accompanied by reduced side effects. In this study, we examine in more detail the cytokinetic parameters of human colon adenocarcinoma HT-29 cells pre-treated for 48 or 24h with LOX inhibitor MK-886, followed by PDT induced by hypericin. Based on MTT assay the concentrations of both agents (MK-886 and hypericin) with relatively slight (non-significant) cytotoxic effects were selected. These concentrations were used for combined treatment, where MTT response, total cell number, floating cells quantification, viability, cell cycle progression and DNA synthesis were detected. Hoechst/PI staining, PARP fragmentation and mitochondrial membrane potential (MMP) were evaluated to determine the extent of apoptosis. While MK-886 alone caused mainly necrosis, 48h pre-treatment of cells with MK-886 followed by PDT with hypericin clearly shifted the type of cell death to apoptosis. PDT with hypericin alone caused apoptosis in 19% of the cell population. Some combined modalities significantly potentiated the apoptotic effect (31% of apoptotic cells; 2.5microM MK-886/0.1microM hypericin), i.e., by 60% more than after single treatment with hypericin. Increased apoptosis was confirmed by PARP (116kDa) cleavage to characteristic 89kDa fragments and changes in MMP. Increasing concentration of MK-886 was accompanied by massive changes in the cell cycle progression. Combined treatment with lower concentrations of MK-886 and hypericin increased accumulation of cells in the S phase, accompanied by inhibition of DNA synthesis. Increasing concentration of MK-886 in this combination caused the opposite effect, manifesting significant accumulation of cells in the G0/G1 phase. More pronounced effects were observed after the 48h pre-treatment schedule. This anti-proliferative effect was confirmed by BrdU incorporation. These results indicate that combined treatment involving PDT and LOX inhibitor MK-886 may improve the therapeutic effectiveness of PDT.

Hypericin-mediated photocytotoxic effect on HT-29 adenocarcinoma cells is reduced by light fractionation with longer dark pause between two unequal light doses.

The present study demonstrates the in vitro effect of hypericin-mediated PDT with fractionated light delivery. Cells were photosensitized with unequal light fractions separated by dark intervals (1 or 6 h). We compared the changes in viability, cell number, survival, apoptosis and cell cycle on HT-29 cells irradiated with a single light dose (12 J/cm(2)) to the fractionated light delivery (1 + 11 J/cm(2)) 24 and 48 h after photodynamic treatment. We found that a fractionated light regime with a longer dark period resulted in a decrease of hypericin cytotoxicity. Both cell number and survival were higher after light sensitization with a 6-h dark interval. DNA fragmentation occurred after a single light-dose application, but in contrast no apoptotic DNA formation was detected with a 6-h dark pause. After fractionation the percentage of cells in the G1 phase of the cell cycle was increased, while the proportion of cells in the G2 phase decreased as compared to a single light-dose application, i.e. both percentage of cells in the G1 and G2 phase of the cell cycle were near control levels. We presume that the longer dark interval after the irradiation of cells by first light dose makes them resistant to the effect of the second illumination. These findings confirm that the light application scheme together with other photodynamic protocol components is crucial for the photocytotoxicity of hypericin.