Complex Alterations of Fatty Acid Metabolism and Phospholipidome Uncovered in Isolated Colon Cancer Epithelial Cells.

The development of colon cancer, one of the most common malignancies, is accompanied with numerous lipid alterations. However, analyses of whole tumor samples may not always provide an accurate description of specific changes occurring directly in tumor epithelial cells. Here, we analyzed in detail the phospholipid (PL), lysophospholipid (lysoPL), and fatty acid (FA) profiles of purified EpCAM+ cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients. We found that a number of FAs increased significantly in isolated tumor cells, which also included a number of long polyunsaturated FAs. Higher levels of FAs were associated with increased expression of FA synthesis genes, as well as with altered expression of enzymes involved in FA elongation and desaturation, including particularly fatty acid synthase, stearoyl-CoA desaturase, fatty acid desaturase 2 and ELOVL5 fatty acid elongase 5 We identified significant changes in ratios of specific lysoPLs and corresponding PLs. A number of lysophosphatidylcholine and lysophosphatidylethanolamine species, containing long-chain and very-long chain FAs, often with high numbers of double bonds, were significantly upregulated in tumor cells. Increased de novo synthesis of very long-chain FAs, or, altered uptake or incorporation of these FAs into specific lysoPLs in tumor cells, may thus contribute to reprogramming of cellular phospholipidome and membrane alterations observed in colon cancer.

Colon Cancer and Perturbations of the Sphingolipid Metabolism.

The development and progression of colorectal cancer (CRC), a major cause of cancer-related death in the western world, is accompanied with alterations of sphingolipid (SL) composition in colon tumors. A number of enzymes involved in the SL metabolism have been found to be deregulated in human colon tumors, in experimental rodent studies, and in human colon cancer cells in vitro. Therefore, the enzymatic pathways that modulate SL levels have received a significant attention, due to their possible contribution to CRC development, or as potential therapeutic targets. Many of these enzymes are associated with an increased sphingosine-1-phosphate/ceramide ratio, which is in turn linked with increased colon cancer cell survival, proliferation and cancer progression. Nevertheless, more attention should also be paid to the more complex SLs, including specific glycosphingolipids, such as lactosylceramides, which can be also deregulated during CRC development. In this review, we focus on the potential roles of individual SLs/SL metabolism enzymes in colon cancer, as well as on the pros and cons of employing the current in vitro models of colon cancer cells for lipidomic studies investigating the SL metabolism in CRC.

Butyrate and docosahexaenoic acid interact in alterations of specific lipid classes in differentiating colon cancer cells.

Docosahexaenoic acid (DHA) and sodium butyrate (NaBt) exhibit a number of interactive effects on colon cancer cell growth, differentiation, or apoptosis; however, the molecular mechanisms responsible for these interactions and their impact on cellular lipidome are still not fully clear. Here, we show that both dietary agents together induce dynamic alterations of lipid metabolism, specific cellular lipid classes, and fatty acid composition. In HT-29 cell line, a model of differentiating colon carcinoma cells, NaBt supported incorporation of free DHA into non-polar lipids and their accumulation in cytoplasmic lipid droplets. DHA itself was not incorporated into sphingolipids; however, it significantly altered representation of individual ceramide (Cer) classes, in particular in combination with NaBt (DHA/NaBt). We observed altered expression of enzymes involved in Cer metabolism in cells treated with NaBt or DHA/NaBt, and exogenous Cer 16:0 was found to promote induction of apoptosis in differentiating HT-29 cells. NaBt, together with DHA, increased n-3 fatty acid synthesis and attenuated metabolism of monounsaturated fatty acids. Finally, DHA and/or NaBt altered expression of proteins involved in synthesis of fatty acids, including elongase 5, stearoyl CoA desaturase 1, or fatty acid synthase, with NaBt increasing expression of caveolin-1 and CD36 transporter, which may further promote DHA incorporation and its impact on cellular lipidome. In conclusion, our results indicate that interactions of DHA and NaBt exert complex changes in cellular lipidome, which may contribute to the alterations of colon cancer cell differentiation/apoptotic responses. The present data extend our knowledge about the nature of interactive effects of dietary fatty acids.

Dietary fatty acids specifically modulate phospholipid pattern in colon cells with distinct differentiation capacities.

PURPOSE: Although beneficial effects of the dietary n-3 docosahexaenoic acid (DHA) or butyrate in colon carcinogenesis have been implicated, the mechanisms of their action are not fully clear. Here, we investigated modulations of composition of individual phospholipid (PL) classes, with a particular emphasis on cardiolipins (CLs), in colon cells treated with DHA, sodium butyrate (NaBt), or their combination (DHA/NaBt), and we evaluated possible associations between lipid changes and cell fate after fatty acid treatment. METHODS: In two distinct human colon cell models, foetal colon (FHC) and adenocarcinoma (HCT-116) cells, we compared patterns and composition of individual PL classes following the fatty acid treatment by HPLC-MS/MS. In parallel, we measured the parameters reflecting cell proliferation, differentiation and death. RESULTS: In FHC cells, NaBt induced primarily differentiation, while co-treatment with DHA shifted their response towards cell death. In contrast, NaBt induced apoptosis in HCT-116 cells, which was not further affected by DHA. DHA was incorporated in all main PL types, increasing their unsaturation, while NaBt did not additionally modulate these effects in either cell model. Nevertheless, we identified an unusually wide range of CL species to be highly increased by NaBt and particularly by DHA/NaBt, and these effects were more pronounced in HCT-116 cells. DHA and DHA/NaBt enhanced levels of high molecular weight and more unsaturated CL species, containing DHA, which was specific for either differentiation or apoptotic responses. CONCLUSIONS: We identified a wide range of CL species in the colon cells which composition was significantly modified after DHA and NaBt treatment. These specific CL modulations might contribute to distinct cellular differentiation or apoptotic responses.

Butyrate alters expression of cytochrome P450 1A1 and metabolism of benzo[a]pyrene via its histone deacetylase activity in colon epithelial cell models.

Butyrate, a short-chain fatty acid produced by fermentation of dietary fiber, is an important regulator of colonic epithelium homeostasis. In this study, we investigated the impact of this histone deacetylase (HDAC) inhibitor on expression/activity of cytochrome P450 family 1 (CYP1) and on metabolism of carcinogenic polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP), in colon epithelial cells. Sodium butyrate (NaBt) strongly potentiated the BaP-induced expression of CYP1A1 in human colon carcinoma HCT116 cells. It also co-stimulated the 7-ethoxyresorufin-O-deethylase (EROD) activity induced by the 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical ligand of the aryl hydrocarbon receptor. Up-regulation of CYP1A1 expression/activity corresponded with an enhanced metabolism of BaP and formation of covalent DNA adducts. NaBt significantly potentiated CYP1A1 induction and/or metabolic activation of BaP also in other human colon cell models, colon adenoma AA/C1 cells, colon carcinoma HT-29 cells, or in NCM460D cell line derived from normal colon mucosa. Our results suggest that the effects of NaBt were due to its impact on histone acetylation, because additional HDAC inhibitors (trichostatin A and suberanilohydroxamic acid) likewise increased both the induction of EROD activity and formation of covalent DNA adducts. NaBt-induced acetylation of histone H3 (at Lys14) and histone H4 (at Lys16), two histone modifications modulated during activation of CYP1A1 transcription, and it reduced binding of HDAC1 to the enhancer region of CYP1A1 gene. This in vitro study suggests that butyrate, through modulation of histone acetylation, may potentiate induction of CYP1A1 expression, which might in turn alter the metabolism of BaP within colon epithelial cells.

DHA-mediated enhancement of TRAIL-induced apoptosis in colon cancer cells is associated with engagement of mitochondria and specific alterations in sphingolipid metabolism.

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid present in fish oil, may exert cytotoxic and/or cytostatic effects on colon cancer cells when applied individually or in combination with some anticancer drugs. Here we demonstrate a selective ability of subtoxic doses of DHA to enhance antiproliferative and apoptotic effects of clinically useful cytokine TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in cancer but not normal human colon cells. DHA-mediated stimulation of TRAIL-induced apoptosis was associated with extensive engagement of mitochondrial pathway (Bax/Bak activation, drop of mitochondrial membrane potential, cytochrome c release), activation of endoplasmic reticulum stress response (CHOP upregulation, changes in PERK level), decrease of cellular inhibitor of apoptosis protein (XIAP, cIAP1) levels and significant changes in sphingolipid metabolism (intracellular levels of ceramides, hexosyl ceramides, sphingomyelines, sphingosines; HPLC/MS/MS). Interestingly, we found significant differences in representation of various classes of ceramides (especially C16:0, C24:1) between the cancer and normal colon cells treated with DHA and TRAIL, and suggested their potential role in the regulation of the cell response to the drug combination. These study outcomes highlight the potential of DHA for a new combination therapy with TRAIL for selective elimination of colon cancer cells via simultaneous targeting of multiple steps in apoptotic pathways.

Interaction of dietary fatty acids with tumour necrosis factor family cytokines during colon inflammation and cancer.

Intestinal homeostasis is precisely regulated by a number of endogenous regulatory molecules but significantly influenced by dietary compounds. Malfunction of this system may result in chronic inflammation and cancer. Dietary essential n-3 polyunsaturated fatty acids (PUFAs) and short-chain fatty acid butyrate produced from fibre display anti-inflammatory and anticancer activities. Both compounds were shown to modulate the production and activities of TNF family cytokines. Cytokines from the TNF family (TNF- α, TRAIL, and FasL) have potent inflammatory activities and can also regulate apoptosis, which plays an important role in cancer development. The results of our own research showed enhancement of apoptosis in colon cancer cells by a combination of either docosahexaenoic acid (DHA) or butyrate with TNF family cytokines, especially by promotion of the mitochondrial apoptotic pathway and modulation of NF κ B activity. This review is focused mainly on the interaction of dietary PUFAs and butyrate with these cytokines during colon inflammation and cancer development. We summarised recent knowledge about the cellular and molecular mechanisms involved in such effects and outcomes for intestinal cell behaviour and pathologies. Finally, the possible application for the prevention and therapy of colon inflammation and cancer is also outlined.

Cisplatin and a potent platinum(IV) complex-mediated enhancement of TRAIL-induced cancer cells killing is associated with modulation of upstream events in the extrinsic apoptotic pathway.

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can selectively trigger apoptosis in various cancer cell types. However, many cancer cells are resistant to death receptor-mediated apoptosis. Combination therapy with platinum complexes may affect TRAIL-induced signaling via modulation of various steps in apoptotic pathways. Here, we show that cisplatin or a more potent platinum(IV) complex LA-12 used in 20-fold lower concentration enhanced killing effects of TRAIL in human colon and prostate cancer cell lines via stimulation of caspase activity and overall apoptosis. Both platinum complexes increased DR5 surface expression in colon cancer cells. Small interfering RNA-mediated DR5 silencing rescued cells from sensitizing effects of platinum drugs on TRAIL-induced caspase-8 activation and apoptosis, showing the functional importance of DR5 in the effects observed. In addition, both cisplatin and LA-12 triggered the relocalization of DR4 and DR5 receptors to lipid rafts and accelerated internalization of TRAIL, which may also affect TRAIL signaling. Collectively, modulations of the initial steps of the extrinsic apoptotic pathway at the level of DR5 and plasma membrane are important for sensitization of colon and prostate cancer cells to TRAIL-induced apoptosis mediated by LA-12 and cisplatin.

Detachment-mediated resistance to TRAIL-induced apoptosis is associated with stimulation of the PI3K/Akt pathway in fetal and adenocarcinoma epithelial colon cells.

The resistance of transformed epithelial cells to a detachment-induced apoptosis (anoikis) can significantly affect their susceptibility to anticancer therapy. We showed that detachment of both fetal (FHC) and adenocarcinoma (HT-29) human colon epithelial cells resulted in the activation of the pro-survival Akt pathway, and significant changes in integrin-linked kinase (ILK) and focal adhesive kinase (FAK) phosphorylation. We demonstrated a detachment-induced and PI3K/Akt-mediated resistance to apoptotic effects of TRAIL, which was not associated with any changes in the cell surface TRAIL death receptor levels. Instead, a modulation of downstream intracellular signaling events was suggested to be involved. Our results may have important implications for optimization of new strategies in treatment of cancers at different stages of development.

Lower sensitivity of FHC fetal colon epithelial cells to photodynamic therapy compared to HT-29 colon adenocarcinoma cells despite higher intracellular accumulation of hypericin.

Preferential uptake of photosensitizer by tumour tissue is an elementary prerequisite of effective and successful photodynamic therapy (PDT). Therefore intracellular concentration of photosensitizer is one of the limiting factors affecting PDT efficiency. Hypericin (HY) has found applications in photodynamic diagnostics solely due to its high specificity for tumour cells and tissues. However, here we suggest that not only HY uptake, but importantly also the cell ability to manage oxidative stress induced by HY-PDT can be important decisive factors finally affecting the cell death response. We showed that despite the higher accumulation of HY in FHC human fetal colon epithelial cells compared to HT-29 colon adenocarcinoma cells, the cytotoxic effects of this photosensitizer were more pronounced in the latter cell line, and this was associated with enhanced accumulation of HY-PDT-induced reactive oxygen species (ROS).

Differences in TRAIL-induced changes of Mcl-1 expression among distinct human colon epithelial cell lines.

In addition to its ability to act as a promising inducer of tumor-specific cell death, TRAIL has also been shown to stimulate signaling pathways leading to cancer cell survival. We examined the changes of anti-apoptotic Mcl-1 protein level following TRAIL treatment of human cell lines representing different stages of colon carcinogenesis-adenocarcinoma (HT-29, HCT116) or secondary metastasis (SW620), together with cell line derived from human fetal colon (FHC). While TRAIL was capable of triggering an anti-apoptotic signaling leading to significant early ERK-mediated transcriptional up-regulation of Mcl-1 in selected colon adenocarcinoma cell lines, none or very limited effects were demonstrated in cell lines derived from colon lymph node metastasis or fetal colon, respectively. We demonstrated an immediate impact of Mcl-1 protein level manipulations on the course of early acute apoptotic response of colon adenocarcinoma cells to TRAIL. It is therefore essential to consider the dynamics of modulation of Mcl-1 level and the balance between TRAIL-induced pro- and anti-apoptotic pathways when predicting the response of cells in different stages of cancer development, and designing the anticancer therapy using TRAIL.

Phospholipid profiling enables to discriminate tumor- and non-tumor-derived human colon epithelial cells: Phospholipidome similarities and differences in colon cancer cell lines and in patient-derived cell samples.

Identification of changes of phospholipid (PL) composition occurring during colorectal cancer (CRC) development may help us to better understand their roles in CRC cells. Here, we used LC-MS/MS-based PL profiling of cell lines derived from normal colon mucosa, or isolated at distinct stages of CRC development, in order to study alterations of PL species potentially linked with cell transformation. We found that a detailed evaluation of phosphatidylinositol (PI) and phosphatidylserine (PS) classes allowed us to cluster the studied epithelial cell lines according to their origin: i) cells originally derived from normal colon tissue (NCM460, FHC); ii) cell lines derived from colon adenoma or less advanced differentiating adenocarcinoma cells (AA/C1, HT-29); or, iii) cells obtained by in vitro transformation of adenoma cells and advanced colon adenocarcinoma cells (HCT-116, AA/C1/SB10, SW480, SW620). Although we tentatively identified several PS and PI species contributing to cell line clustering, full PI and PS profiles appeared to be a key to the successful cell line discrimination. In parallel, we compared PL composition of primary epithelial (EpCAM-positive) cells, isolated from tumor and adjacent non-tumor tissues of colon cancer patients, with PL profiles of cell lines derived from normal colon mucosa (NCM460) and from colon adenocarcinoma (HCT-116, SW480) cells, respectively. In general, higher total levels of all PL classes were observed in tumor cells. The overall PL profiles of the cell lines, when compared with the respective patient-derived cells, exhibited similarities. Nevertheless, there were also some notable differences in levels of individual PL species. This indicated that epithelial cell lines, derived either from normal colon tissue or from CRC cells, could be employed as models for functional lipidomic analyses of colon cells, albeit with some caution. The biological significance of the observed PL deregulation, or their potential links with specific CRC stages, deserve further investigation.

5-Lipoxygenase inhibitors potentiate 1alpha,25-dihydroxyvitamin D3-induced monocytic differentiation by activating p38 MAPK pathway.

The treatment of human promyelocytic leukemia cell lines HL-60, and to some extent NB-4, with 1alpha,25-dihydroxyvitamin D(3) (VD3) induces differentiation toward the monocytic/macrophage lineage, demonstrated by the increased expression of CD11b and CD14, and the production of opsonized zymosan particles (OZP)-stimulated reactive oxygen species (ROS). Moreover, in more sensitive HL-60 cells, increased expression of 5-lipoxygenase (5-LPO), Mcl-1, IkappaB, and c-Jun, accompanied by the activation of p38 MAPK, was detected. These VD3 effects on HL-60 cell differentiation were significantly potentiated by 5-LPO inhibitors MK-886 and AA-861 and were inverted by SB202190 (SB), a p38 MAPK inhibitor. The inhibition of differentiation by SB was demonstrated by a reduction of CD14 expression and by a decrease in OZP-activated ROS production. These results indicated that p38 MAPK pathway is involved in 5-LPO inhibitors-dependent potentiation of VD3-induced monocytic differentiation.

Monocytic differentiation of leukemic HL-60 cells induced by co-treatment with TNF-alpha and MK886 requires activation of pro-apoptotic machinery.

The block of hematopoietic differentiation program in acute myeloid leukemia cells can be overcome by differentiating agent like retinoic acid, but it has several side effects. A study of other differentiation signaling pathways is therefore useful to predict potential targets of anti-leukemic therapy. We demonstrated previously that the co-treatment of HL-60 cells with Tumor necrosis factor-alpha (TNF-alpha) (1 ng/mL) and inhibitor of 5-lipoxygenase MK886 (5 microm) potentiated both monocytic differentiation and apoptosis. In this study, we detected enhanced activation of three main types of mitogen-activated protein kinases (MAPKs) (p38, c-Jun amino-terminal kinase [JNK], extracellular signal-regulated kinase [ERK]), so we assessed their role in differentiation using appropriate pharmacologic inhibitors. The inhibition of pro-apoptotic MAPKs (p38 and JNK) suppressed the effect of MK886 + TNF-alpha co-treatment. On the other hand, down-regulation of pro-survival ERK pathway led to increased differentiation. Those effects were accompanied by increased activation of caspases in cells treated by MK886 + TNF-alpha. Pan-caspase inhibitor ZVAD-fmk significantly decreased both number of apoptotic and differentiated cells. The same effect was observed after inhibition of caspase 9, but not caspase 3 and 8. To conclude, we evidenced that the activation of apoptotic processes and pathways supporting apoptosis (p38 and JNK MAPKs) is required for the monocytic differentiation of HL-60 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.

Butyrate interacts with benzo[a]pyrene to alter expression and activities of xenobiotic metabolizing enzymes involved in metabolism of carcinogens within colon epithelial cell models.

Butyrate helps to maintain colon homeostasis and exhibits chemopreventive effects in colon epithelium. We examined the interactive effects of butyrate and benzo[a]pyrene (BaP), dietary carcinogen, in regulation of expression of a panel of phase I and II xenobiotic metabolizing enzymes (XMEs) in human colon cells. In human colon carcinoma HCT-116 and HT-29 cell lines, butyrate alone increased mRNA levels of some enzymes, such as N-acetyltransferases (in particular NAT2). In combination with BaP, butyrate potentiated induction of cytochrome P450 family 1 enzymes (CYP1A1), aldo-keto reductases (AKR1C1) or UDP-glucuronosyltransferases (UGT1A1). There were some notable differences between cell lines, as butyrate potentiated induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) and UGT1A4 only in HCT-116 cells, and it even repressed AKR1C3 induction in HT-29 cells. Butyrate also promoted induction of CYP1, NQO1, NAT2, UGT1A1 or UGT1A4 in human colon Caco-2 cells, in a differentiation-dependent manner. Differentiated Caco-2 cells exhibited a higher inducibility of selected XME genes than undifferentiated cells. Butyrate increased induction of enzymatic activities of NATs, NQO1 and UGTs by BaP in HCT-116 and HT29 cells, whereas in differentiated Caco-2 cells it helped to increase only enzymatic activity of NQO1 and UGTs. Together, the present data suggest that butyrate may modulate expression/activities of several enzymes involved in metabolism of carcinogens in colon. In some cases (NAT2, UGT1 A1), this was linked to inhibition of histone deacetylases (HDAC), as confirmed by using HDAC inhibitor trichostatin A. These results may have implications for our understanding of the role of butyrate in regulation of XMEs and carcinogen metabolism in colon.

n-3 Polyunsaturated fatty acids alter benzo[a]pyrene metabolism and genotoxicity in human colon epithelial cell models.

Dietary carcinogens, such as benzo[a]pyrene (BaP), are suspected to contribute to colorectal cancer development. n-3 Polyunsaturated fatty acids (PUFAs) decrease colorectal cancer risk in individuals consuming diets rich in PUFAs. Here, we investigated the impact of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid on metabolism and genotoxicity of BaP in human cell models derived from the colon: HT-29 and HCT-116 cell lines. Both PUFAs reduced levels of excreted BaP metabolites, in particular BaP-tetrols and hydroxylated BaP metabolites, as well as formation of DNA adducts in HT-29 and HCT-116 cells. However, EPA appeared to be a more potent inhibitor of formation of some intracellular BaP metabolites, including BaP-7,8-dihydrodiol. EPA also reduced phosphorylation of histone H2AX (Ser139) in HT-29 cells, which indicated that it may reduce further forms of DNA damage, including DNA double strand breaks. Both PUFAs inhibited induction of CYP1 activity in colon cells determined as 7-ethoxyresorufin-O-deethylase (EROD); this was at least partly linked with inhibition of induction of CYP1A1, 1A2 and 1B1 mRNAs. The downregulation and/or inhibition of CYP1 enzymes by PUFAs could thus alter metabolism and reduce genotoxicity of BaP in human colon cells, which might contribute to known chemopreventive effects of PUFAs in colon epithelium.

Response of normal and colon cancer epithelial cells to TNF-family apoptotic inducers.

We compared the response of normal (FHC) and cancer (HT-29) human colon epithelial cells to the important apoptotic inducers TNF-alpha, anti-Fas antibody and TNF-related apoptosis inducing ligand (TRAIL). The two cell lines did not respond to TNF-alpha (15 ng/ml), expressed a limited sensitivity to anti-Fas antibody (200 ng/ml) and a different response to TRAIL (100 ng/ml). We studied apoptosis with regard to the changes at the receptor level (DR, DcR and FLIP) and at the level of mitochondria (Bid protein cleavage, Apo2.7 protein expression and caspase-9 activation). Two different approaches were used to sensitize the cells to TRAIL-induced apoptosis: inhibition of protein synthesis (cycloheximide, CHX) and inhibition of the pro-survival MEK/ERK pathway (U0126). While the two cell lines were markedly sensitized to all three TNF family members by CHX, a different degree of response (especially for TRAIL) was obtained when inhibition of the MEK/ERK pathway was achieved. TRAIL-induced apoptosis was significantly enhanced by U0126 co-treatment in the HT-29 cells, but not in the FHC cells. The most significant differences between the HT-29 and FHC cells co-treated with TRAIL and U0126 were demonstrated with regard to the involvement of the mitochondrial apoptotic pathway, suggesting its importance in the regulation of cell sensitivity to the TRAIL-induced apoptosis.

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.