Combination of photoactive hypericin and Manumycin A exerts multiple anticancer effects on oxaliplatin-resistant colorectal cells.

The use of natural products as chemotherapeutic agents and tools for manipulation of apoptosis represent an attractive therapeutic concept. In this study, we investigated the anticancer activities of a combination of two natural compounds with different origin, hypericin (plant product) in its photoactive state and Manumycin A (yeast product) and explored the underlying mechanisms of their pro-apoptotic action using an oxaliplatin-resistant variant of human colon adenocarcinoma cell line HT-29-OxR as the experimental model. CCK-8 assay was performed to evaluate the cytotoxicity of the drugs. CalcuSyn software was used to identify the type of interaction between the two agents. BrdU incorporation assay and colony forming assay were performed to study the short- and long-term proliferation of cells. To evaluate the ability of the drug combination to induce apoptosis, PARP p85 fragment was detected using the ELISA method. Changes in apoptosis-related proteins were examined by immunoassays. Our results showed that a synergistic combination of photoactive hypericin and Manumycin A decreased viability, inhibited both short- and long-term cell proliferation, decreased levels of IAPs proteins (cIAP1, cIAP2, XIAP and survivin), induced an apoptotic PARP cleavage associated with decline in procaspase-3 level, promoted phagocytosis of cancer cells, and restored chemosensitivity to oxaliplatin.

The potential of hypericin and hyperforin for antiadhesion therapy to prevent metastasis of parental and oxaliplatin-resistant human adenocarcinoma cells (HT-29).

Cancer cells disseminate to other parts of the body during metastasis through the process of intravasation. The hypericin and hyperforin effect has been described to understand the signal mechanisms that stimulate or stunt cancer cell sprouting to metastasis on colon adenocarcinoma cells HT-29 and its resistant form HT-29-OxR. We focused on the key points of adhesion proteins (cadherin, integrin, selectin and syndecan) and also proteins participating in or contributing to the process of cancer cell migration and adhesion through genes expression and proteins levels. Treatment effects were identified as a consequence of decreased cell adhesion, changes of expression in the adhesive proteins as well as basal membrane degradation associated with changes in the expression of matrix proteinases and in their activity. Finally, the cells affected by hypericin or hyperforin were evaluated by monitoring the cancer cell adhesion properties and proliferation processes. Supplementary Fig. (Supplemental digital content 1, http://links.lww.com/ACD/A267).

Photoactivated hypericin increases the expression of SOD-2 and makes MCF-7 cells resistant to photodynamic therapy.

Photoactivated hypericin increased production of reactive oxygen species in human breast adenocarcinoma MCF-7 as well as in MDA-MB-231 cells 1h after photodynamic therapy. On the other hand, reactive oxygen species dropped 3h after photodynamic therapy with hypericin, but only in MCF-7 cells, whereas in MDA-MB-231 cells remained elevated. The difference in the dynamics of reactive oxygen species after hypericin activation was related to increased activity of SOD-2 in MCF-7 cells compared to MDA-MB-231 cells. Indeed, photodynamic therapy with hypericin significantly increased SOD-2 activity in MCF-7 cells, but only slightly in MDA-MB-231 cells. In this regard, SOD-2 activity correlated well with enhanced both mRNA expression as well as SOD-2 protein level in MCF-7 cells. The role of SOD-2 in the resistance of MCF-7 cells to photodynamic therapy with hypericin was monitored using SOD-2 inhibitor - 2-methoxyestradiol. Interestingly, the combination of photodynamic therapy with hypericin and methoxyestradiol sensitized MCF-7 cells to photodynamic therapy and significantly reduced its clonogenic ability. Furthermore, methoxyestradiol potentiated the activation of caspase 3/7 and apoptosis induced by photodynamic therapy with hypericin.

Antitumor effect of the combination of manumycin A and Immodin is associated with antiplatelet activity and increased granulocyte tumor infiltration in a 4T1 breast tumor model.

Manumycin A is a natural antibiotic isolated from Streptomyces parvulus with broad range of biological activities including antineoplastic activity in several in vitro and in vivo cancer models. Immodin [dialyzable leukocyte extract (DLE)] is a dialysate released from disintegrated blood leukocytes of healthy donors which exerts immunonormalizing effects on cell-mediated immune responses. The aim of the present study was to explore the antitumor potential of the combination of manumycin A and Immodin in an experimental breast cancer model. Experiments were carried using a 4T1 tumor-bearing BALB/c mouse model. Survival analysis, tumor growth, hematological and biochemical profiles, leukocyte differential, phagocytic activity of leukocytes and histology of the primary tumor were examined. The combination treatment suppressed the tumor growth and prolonged the survival of tumor-bearing mice, decreased the number of monocytes, plateletes and plateletcrit in peripheral blood of the tumor-bearing mice and increased the infiltration of neutrophils and eosinophils in the primary tumor. Moreover, individual therapies enhanced the phagocytic activity of monocytes and neutrophils. These findings demonstrate the antitumor effect of the combination of manumycin A and Immodin in 4T1 tumor-bearing mice associated with strong antiplatelet activity and innate immunity activation.

Far-western blotting as a solution to the non-specificity of the anti-erythropoietin receptor antibody.

The erythropoietin receptor (EpoR) is a member of the cytokine receptor family. The interaction between erythropoietin (Epo) and EpoR is important for the production and maturation of erythroid cells, resulting in the stimulation of hematopoiesis. The fact that EpoR was also detected in neoplastic cells has opened the question about the relevance of anemia treatment with recombinant Epo in cancer patients. Numerous studies have reported pro-stimulating and anti-apoptotic effects of Epo in cancer cells, thus demonstrating EpoR functionality in these cells. By contrast, a previous study claims the absence of EpoR in tumor cells. This apparent discrepancy is based, according to certain authors, on the use of non-specific anti-EpoR antibodies. With the aim of bypassing the direct detection of EpoR with an anti-EpoR antibody, the present authors propose a far-western blot methodology, which in addition, confirms the interaction of Epo with EpoR. Applying this technique, the presence of EpoR and its interaction with Epo in human ovarian adenocarcinoma A2780 and normal human umbilical vein endothelial cells was confirmed. Furthermore, modified immunoprecipitation of EpoR followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis confirmed a 57 kDa protein as a human Epo-interacting protein in both cell lines.

Enhanced antiproliferative and apoptotic response of HT-29 adenocarcinoma cells to combination of photoactivated hypericin and farnesyltransferase inhibitor manumycin A.

Several photodynamically-active substances and farnesyltransferase inhibitors are currently being investigated as promising anticancer drugs. In this study, the combined effect of hypericin (the photodynamically-active pigment from Hypericum perforatum) and selective farnesyltransferase inhibitor manumycin (manumycin A; the selective farnesyltransferase inhibitor from Streptomyces parvulus) on HT-29 adenocarcinoma cells was examined. We found that the combination treatment of cells with photoactivated hypericin and manumycin resulted in enhanced antiproliferative and apoptotic response compared to the effect of single treatments. This was associated with increased suppression of clonogenic growth, S phase cell cycle arrest, elevated caspase-3/7 activity and time-dependent total cleavage of procaspase-3 and lamin B, cleavage of p21Bax into p18Bax and massive PARP cleavage. Moreover, we found that the apoptosis-inducing factor is implicated in signaling events triggered by photoactivated hypericin. Our results showed the relocalization of apoptosis-inducing factor (AIF) to the nuclei after hypericin treatment. In addition, we discovered that not only manumycin but also photoactivated hypericin induced the reduction of total Ras protein level. Manumycin decreased the amount of farnesylated Ras, and the combination treatment decreased the amount of both farnesylated and non-farnesylated Ras protein more dramatically. The present findings indicate that the inhibition of Ras processing may be the determining factor for enhancing the antiproliferative and apoptotic effects of combination treatment on HT-29 cells.

Drug efflux transporters, MRP1 and BCRP, affect the outcome of hypericin-mediated photodynamic therapy in HT-29 adenocarcinoma cells.

Photodynamic therapy (PDT) is a flexible multi-target therapeutic approach. One of the main requirements of successful PDT is sufficient intracellular concentration of an applicable photosensitizer. Mechanisms of anticancer drug elimination by tumour cells are mostly linked to the elevated expression and activity of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), breast cancer resistance protein (BCRP) and P450 monooxygenases. The interaction of hypericin with this cell drug-defence system is still unclear. We report here for the first time increased activity of MRP1 and BCRP in HT-29 colon cancer cells treated with hypericin per se. On the contrary, pre-treatment with proadifen (SKF525A) affected the function of MRP1 and BCRP leading to increased hypericin content, which might indicate a possible link between proadifen and these ABC transporter proteins. Subsequent enhanced intracellular oxidative stress was accompanied by loss of mitochondrial membrane potential, activation of caspase-9 and -3, PARP cleavage and onset of apoptosis. In conclusion, our study suggests that drug efflux transporters MRP1 and BCRP affect the pharmacokinetics of hypericin in HT-29 colon adenocarcinoma cells, and the action of hypericin-mediated PDT (HY-PDT) should be modulated by pre-treatment with their specific inhibitors.

The role of p53 in the efficiency of photodynamic therapy with hypericin and subsequent long-term survival of colon cancer cells.

Photodynamic therapy with hypericin (HY-PDT) is known as an efficient modality for treatment of various cancerous and non-cancerous diseases. Although the role of p53 protein in cell death signaling is well established, relatively little is known of its impact on the efficiency of HY-PDT. Comparison of sensitivity and long-term survival of p53-null versus wt-p53-expressing HCT-116 cells is reported here. The lack of p53 function did not affect cell proliferation or attenuate the initial phases of programmed cell death. However, analyses of apoptosis in the final stages revealed suppression of its incidence and delayed activation of caspase-3 in p53-null cells. Significantly higher clonogenic ability, especially in hypoxia, was identified in the case of p53-null cells. Induction of Mcl-1 and Bax levels were more prominent in wt-pt53 cells. Interestingly, the level of Bcl-2 did not react to HY-PDT at all, in both cell lines. Bringing the evidence together, we prove that despite insignificant impact on overall toxicity, expression of p53 affects the clonogenic efficiency of HCT-116 cells. Since destruction of tumor tissue and its vascular system by PDT tends to lead to hypoxia, superior survival of p53-deficient tumor cells under given conditions might result in recurrence of cancer diseases.

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.

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.