ABCB1 Amplicon Contains Cyclic AMP Response Element-Driven TRIP6 Gene in Taxane-Resistant MCF-7 Breast Cancer Sublines.

A limited number of studies are devoted to regulating TRIP6 expression in cancer. Hence, we aimed to unveil the regulation of TRIP6 expression in MCF-7 breast cancer cells (with high TRIP6 expression) and taxane-resistant MCF-7 sublines (manifesting even higher TRIP6 expression). We found that TRIP6 transcription is regulated primarily by the cyclic AMP response element (CRE) in hypomethylated proximal promoters in both taxane-sensitive and taxane-resistant MCF-7 cells. Furthermore, in taxane-resistant MCF-7 sublines, TRIP6 co-amplification with the neighboring ABCB1 gene, as witnessed by fluorescence in situ hybridization (FISH), led to TRIP6 overexpression. Ultimately, we found high TRIP6 mRNA levels in progesterone receptor-positive breast cancer and samples resected from premenopausal women.

Expression profiles of iron transport molecules along the duodenum.

Duodenal biopsies are considered a suitable source of enterocytes for studies of dietary iron absorption. However, the expression level of molecules involved in iron absorption may vary along the length of duodenum. We aimed to determine whether the expression of molecules involved in the absorption of heme and non-heme iron differs depending on the location in the duodenum. Analysis was performed with samples of duodenal biopsies from 10 individuals with normal iron metabolism. Samples were collected at the following locations: (a) immediately post-bulbar, (b) 1-2 cm below the papilla of Vater and (c) in the distal duodenum. The gene expression was analyzed at the mRNA and protein level using real-time PCR and Western blot analysis. At the mRNA level, significantly different expression of HCP1, DMT1, ferroportin and Zip8 was found at individual positions of duodenum. Position-dependent expression of other molecules, especially of FLVCR1, HMOX1 and HMOX2 was also detected but with no statistical significances. At the protein level, we observed statistically significantly decreasing expression of transporters HCP1, FLVCR1, DMT1, ferroportin, Zip14 and Zip8 with advancing positions of duodenum. Our results are consistent with a gradient of diminishing iron absorption along the duodenum for both heme and non-heme iron.

Iron-dependent apoptosis causes embryotoxicity in inflamed and obese pregnancy.

Iron is essential for a healthy pregnancy, and iron supplementation is nearly universally recommended, regardless of maternal iron status. A signal of potential harm is the U-shaped association between maternal ferritin, a marker of iron stores, and risk of adverse pregnancy outcomes. However, ferritin is also induced by inflammation and may overestimate iron stores during inflammation or infection. In this study, we use mouse models to determine whether maternal iron loading, inflammation, or their interaction cause poor pregnancy outcomes. Only maternal exposure to both iron excess and inflammation, but not either condition alone, causes embryo malformations and demise. Maternal iron excess potentiates embryo injury during both LPS-induced acute inflammation and obesity-induced chronic mild inflammation. The adverse interaction depends on TNFα signaling, causes apoptosis of placental and embryo endothelium, and is prevented by anti-TNFα or antioxidant treatment. Our findings raise important questions about the safety of indiscriminate iron supplementation during pregnancy.

The Role of TRIP6, ABCC3 and CPS1 Expression in Resistance of Ovarian Cancer to Taxanes.

The main problem precluding successful therapy with conventional taxanes is de novo or acquired resistance to taxanes. Therefore, novel experimental taxane derivatives (Stony Brook taxanes; SB-Ts) are synthesized and tested as potential drugs against resistant solid tumors. Recently, we reported alterations in ABCC3, CPS1, and TRIP6 gene expression in a breast cancer cell line resistant to paclitaxel. The present study aimed to investigate gene expression changes of these three candidate molecules in the highly resistant ovarian carcinoma cells in vitro and corresponding in vivo models treated with paclitaxel and new experimental Stony Brook taxanes of the third generation (SB-T-121605 and SB-T-121606). We also addressed their prognostic meaning in ovarian carcinoma patients treated with taxanes. We estimated and observed changes in mRNA and protein profiles of ABCC3, CPS1, and TRIP6 in resistant and sensitive ovarian cancer cells and after the treatment of resistant ovarian cancer models with paclitaxel and Stony Brook taxanes in vitro and in vivo. Combining Stony Brook taxanes with paclitaxel caused downregulation of CPS1 in the paclitaxel-resistant mouse xenograft tumor model in vivo. Moreover, CPS1 overexpression seems to play a role of a prognostic biomarker of epithelial ovarian carcinoma patients' poor survival. ABCC3 was overexpressed in EOC tumors, but after the treatment with taxanes, its up-regulation disappeared. Based on our results, we can suggest ABCC3 and CPS1 for further investigations as potential therapeutic targets in human cancers.

Upregulation of vitamin D-binding protein is associated with changes in insulin production in pancreatic beta-cells exposed to p,p'-DDT and p,p'-DDE.

Persistent organochlorine pollutants (POPs) gradually accumulate in the human organism due to their presence in the environment. Some studies have described a correlation between the level of POPs in the human body and the incidence of diabetes, but we know little about the direct effect of POPs on pancreatic beta-cells. We exposed pancreatic beta-cells INS1E to non-lethal concentrations of p,p'-DDT (1,1'-(2,2,2-Trichloroethane-1,1-diyl)bis(4-chlorobenzene)) and p,p'-DDE (1,1'-(2,2-dichloroethene-1,1-diyl)bis(4-chlorobenzene)) for 1 month, and assessed changes in protein expression and the intracellular insulin level. 2-D electrophoresis revealed 6 proteins with changed expression in cells exposed to p,p'-DDT or p,p'-DDE. One of the detected proteins - vitamin D-binding protein (VDBP) - was upregulated in both cells exposed to p,p'-DDT, and cells exposed to p,p'-DDE. Both exposures to pollutants reduced the intracellular level of insulin mRNA, proinsulin, and insulin monomer; p,p'-DDT also slightly reduced the level of hexameric insulin. Overexpression of VDBP caused by the stable transfection of beta-cells with the gene for VDBP decreased both the proinsulin and hexameric insulin level in beta-cells similarly to the reduction detected in cells exposed to p,p'-DDT. Our data suggest that in the cells exposed to p,p'-DDT and p,p'-DDE, the increased VDBP protein level decreased the proinsulin expression in an unknown mechanism.

Differentially Expressed Mitochondrial Proteins in Human MCF7 Breast Cancer Cells Resistant to Paclitaxel.

Identification of novel proteins with changed expression in resistant cancer cells could be helpful in elucidation mechanisms involved in the development of acquired resistance to paclitaxel. In this study, we carried out a 2D-PAGE using the mitochondrial-enriched fraction from paclitaxel-resistant MCF7/PacR cells compared to original paclitaxel-sensitive MCF7 breast cancer cells. Differentially expressed proteins were identified employing mass spectrometry. We found that lysosomal cathepsin D and mitochondrial abhydrolase-domain containing protein 11 (ABHD11) had decreased expression in MCF7/PacR cells. On the other hand, mitochondrial carbamoyl-phosphate synthetase 1 (CPS1) and ATPase family AAA-domain containing protein 3A and 3B (ATAD3A, ATAD3B) were overexpressed in MCF7/PacR cells. Further, we showed that there was no difference in localization of CPS1 in MCF7 and MCF7/PacR cells. We demonstrated a significant increase in the number of CPS1 positive MCF7/PacR cells, using FACS analysis, compared to the number of CPS1 positive MCF7 cells. Silencing of CPS1 expression by specific siRNA had no significant effect on the resistance of MCF7/PacR cells to paclitaxel. To summarize, we identified several novel proteins of a mitochondrial fraction whose role in acquired resistance to paclitaxel in breast cancer cells should be further assessed.

Stearate-Induced Apoptosis in Human Pancreatic ß-Cells is Associated with Changes in Membrane Protein Expression and These Changes are Inhibited by Oleate.

PURPOSE: Lipotoxicity is implicated in type 2 diabetes pathogenesis. Its molecular mechanisms are not completely understood. The aim of this study is to identify new suspect proteins involved in pancreatic ß-cell death induction by saturated fatty acids and its inhibition by unsaturated fatty acids. EXPERIMENTAL DESIGN: Employing 2DE analysis and subsequent western blot confirmation, the differences in membrane/membrane-associated protein expression in human ß-cell line NES2Y are assessed during cell death induction by stearate and its inhibition by oleate. RESULTS: Induction of apoptosis by stearate is associated with significantly increased levels of Hsp90ß, peroxiredoxin-1, and 14-3-3γ in the membrane fraction of NES2Y cells and significantly decreased levels of annexin A2, annexin A4, and reticulocalbin-2. All these changes are significantly inhibited by oleate co-application. No expression changes are detected after application of stearate together with oleate. Furthermore, the expression of reticulocalbin-2 is significantly decreased after stearate application also in the whole cell lysate. CONCLUSIONS AND CLINICAL RELEVANCE: Several membrane-associated proteins that could be related to pro- and anti-apoptotic signaling initiated by fatty acids in human pancreatic ß-cells are identified. As far as we know, annexin A4, reticulocalbin-2, and 14-3-3γ represent novel molecules related to the effect of fatty acids on ß-cell viability.

The Effect of Hypoxia and Metformin on Fatty Acid Uptake, Storage, and Oxidation in L6 Differentiated Myotubes.

Metabolic impairments associated with obstructive sleep apnea syndrome (OSA) are linked to tissue hypoxia, however, the explanatory molecular and endocrine mechanisms remain unknown. Using gas-permeable cultureware, we studied the chronic effects of mild and severe hypoxia on free fatty acid (FFA) uptake, storage, and oxidation in L6 myotubes under 20, 4, or 1% O2. Additionally, the impact of metformin and the peroxisome proliferator-activated receptor (PPAR) ß/δ agonist, called GW501516, were investigated. Exposure to mild and severe hypoxia reduced FFA uptake by 37 and 32%, respectively, while metformin treatment increased FFA uptake by 39% under mild hypoxia. GW501516 reduced FFA uptake under all conditions. Protein expressions of CD36 (cluster of differentiation 36) and SCL27A4 (solute carrier family 27 fatty acid transporter, member 4) were reduced by 17 and 23% under severe hypoxia. Gene expression of UCP2 (uncoupling protein 2) was reduced by severe hypoxia by 81%. Metformin increased CD36 protein levels by 28% under control conditions and SCL27A4 levels by 56% under mild hypoxia. Intracellular lipids were reduced by mild hypoxia by 18%, while in controls only, metformin administration further reduced intracellular lipids (20% O2) by 36%. Finally, palmitate oxidation was reduced by severe hypoxia, while metformin treatment reduced non-mitochondrial O2 consumption, palmitate oxidation, and proton leak at all O2 levels. Hypoxia directly reduced FFA uptake and intracellular lipids uptake in myotubes, at least partially, due to the reduction in CD36 transporters. Metformin, but not GW501516, can increase FFA uptake and SCL27A4 expression under mild hypoxia. Described effects might contribute to elevated plasma FFA levels and metabolic derangements in OSA.

Substituents at the C3' and C3'N positions are critical for taxanes to overcome acquired resistance of cancer cells to paclitaxel.

We tested the role of substituents at the C3' and C3'N positions of the taxane molecule to identify taxane derivatives capable of overcoming acquired resistance to paclitaxel. Paclitaxel-resistant sublines SK-BR-3/PacR and MCF-7/PacR as well as the original paclitaxel-sensitive breast cancer cell lines SK-BR-3 and MCF-7 were used for testing. Increased expression of the ABCB1 transporter was found to be involved in the acquired resistance. We tested three groups of taxane derivatives: (1) phenyl group at both C3' and C3'N positions, (2) one phenyl at one of the C3' and C3'N positions and a non-aromatic group at the second position, (3) a non-aromatic group at both C3' and C3'N positions. We found that the presence of phenyl groups at both C3' and C3'N positions is associated with low capability of overcoming acquired paclitaxel resistance compared to taxanes containing at least one non-aromatic substituent at the C3' and C3'N positions. The increase in the ATPase activity of ABCB1 transporter after the application of taxanes from the first group was found to be somewhat higher than after the application of taxanes from the third group. Molecular docking studies demonstrated that the docking score was the lowest, i.e. the highest binding affinity, for taxanes from the first group. It was intermediate for taxanes from the second group, and the highest for taxanes from the third group. We conclude that at least one non-aromatic group at the C3' and C3'N positions of the taxane structure, resulting in reduced affinity to the ABCB1 transporter, brings about high capability of taxane to overcome acquired resistance of breast cancer cells to paclitaxel, due to less efficient transport of the taxane compound out of the cancer cells.

Glycol porphyrin derivatives and temoporfin elicit resistance to photodynamic therapy by different mechanisms.

The development of drug resistance is a major problem which often occurs during anticancer chemotherapies. Photodynamic therapy (PDT) has been studied as an alternative treatment modality for drug-resistant tumors, however the question of resistance to PDT and potential cross-resistance with chemotherapy has yet to be fully answered. To investigate the mechanism of resistance to PDT, we developed an in vitro experimental model system in a mouse mammary carcinoma cell line 4T1. We used two ethylene glycol derivatives of tetraphenylporphyrin, and tetraphenylchlorin derivative, temoporfin, as photosensitizers (PS). PDT-resistant clones were obtained by exposure to a set concentration of PS followed by irradiation with increasing light doses. PDT resistance to soluble glycol porphyrins was mediated mainly by increased drug efflux through ABCB1 (P-glycoprotein) as we demonstrated by specific ABCB1 knockdown experiments, which in turn rescued the sensitivity of resistant cells to PDT. In contrast, resistance raised to temoporfin, which is generally more lipophilic than glycol porphyrins, elicited mechanism based on sequestration of the drug to lysosomes. The resistance that is acquired from a particular PS could be overcome by using a different PS, which is not susceptible to the same mechanism(s) of resistance. Elucidation of the underlying mechanisms in various types of resistance might facilitate improvements in PDT treatment design.

Characterization of acquired paclitaxel resistance of breast cancer cells and involvement of ABC transporters.

Development of taxane resistance has become clinically very important issue. The molecular mechanisms underlying the resistance are still unclear. To address this issue, we established paclitaxel-resistant sublines of the SK-BR-3 and MCF-7 breast cancer cell lines that are capable of long-term proliferation in 100nM and 300nM paclitaxel, respectively. Application of these concentrations leads to cell death in the original counterpart cells. Both sublines are cross-resistant to doxorubicin, indicating the presence of the MDR phenotype. Interestingly, resistance in both paclitaxel-resistant sublines is circumvented by the second-generation taxane SB-T-1216. Moreover, we demonstrated that it was not possible to establish sublines of SK-BR-3 and MCF-7 cells resistant to this taxane. It means that at least the tested breast cancer cells are unable to develop resistance to some taxanes. Employing mRNA expression profiling of all known human ABC transporters and subsequent Western blot analysis of the expression of selected transporters, we demonstrated that only the ABCB1/PgP and ABCC3/MRP3 proteins were up-regulated in both paclitaxel-resistant sublines. We found up-regulation of ABCG2/BCRP and ABCC4 proteins only in paclitaxel-resistant SK-BR-3 cells. In paclitaxel-resistant MCF-7 cells, ABCB4/MDR3 and ABCC2/MRP2 proteins were up-regulated. Silencing of ABCB1 expression using specific siRNA increased significantly, but did not completely restore full sensitivity to both paclitaxel and doxorubicin. Thus we showed a key, but not exclusive, role for ABCB1 in mechanisms of paclitaxel resistance. It suggests the involvement of multiple mechanisms in paclitaxel resistance in tested breast cancer cells.

p38 MAPK Is Activated but Does Not Play a Key Role during Apoptosis Induction by Saturated Fatty Acid in Human Pancreatic ß-Cells.

Saturated stearic acid (SA) induces apoptosis in the human pancreatic ß-cells NES2Y. However, the molecular mechanisms involved are unclear. We showed that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway in these cells. Therefore, we tested the role of p38 MAPK signaling pathway activation in apoptosis induction by SA in NES2Y cells. Crosstalk between p38 MAPK pathway activation and accompanying ERK pathway inhibition after SA application was also tested. The inhibition of p38 MAPK expression by siRNA silencing resulted in a decrease in MAPKAPK-2 activation after SA application, but it had no significant effect on cell viability or the level of phosphorylated ERK pathway members. The inhibition of p38 MAPK activity by the specific inhibitor SB202190 resulted in inhibition of MAPKAPK-2 activation and noticeable activation of ERK pathway members after SA treatment but in no significant effect on cell viability. p38 MAPK overexpression by plasmid transfection produced an increase in MAPKAPK-2 activation after SA exposure but no significant influence on cell viability or ERK pathway activation. The activation of p38 MAPK by the specific activator anisomycin resulted in significant activation of MAPKAPK-2. Concerning the effect on cell viability, application of the activator led to apoptosis induction similar to application of SA (PARP cleavage and caspase-7, -8, and -9 activation) and in inhibition of ERK pathway members. We demonstrated that apoptosis-inducing concentrations of SA activate the p38 MAPK signaling pathway and that this activation could be involved in apoptosis induction by SA in the human pancreatic ß-cells NES2Y. However, this involvement does not seem to play a key role. Crosstalk between p38 MAPK pathway activation and ERK pathway inhibition in NES2Y cells seems likely. Thus, the ERK pathway inhibition by p38 MAPK activation does not also seem to be essential for SA-induced apoptosis.

The role of individual caspases in cell death induction by taxanes in breast cancer cells.

BACKGROUND: In previous study we showed that caspase-2 plays the role of an apical caspase in cell death induction by taxanes in breast cancer cells. This study deals with the role of other caspases. We tested breast cancer cell lines SK-BR-3 (functional caspase-3) and MCF-7 (nonfunctional caspase-3). METHODS AND RESULTS: Using western blot analysis we demonstrated the activation of initiator caspase-8 and -9 as well as executioner caspase-6 and -7 in both tested cell lines after application of taxanes (paclitaxel, SB-T-1216) at death-inducing concentrations. Caspase-3 activation was also found in SK-BR-3 cells. Employing specific siRNAs after taxane application, suppression of caspase-3 expression significantly increased the number of surviving SK-BR-3 cells. Inhibition of caspase-7 expression also increased the number of surviving SK-BR-3 and MCF-7 cells. On the other hand, suppression of caspase-8 and caspase-9 expression had no significant effect on cell survival. However, caspase-9 seemed to be involved in the activation of caspase-3 and caspase-7. Caspase-3 and caspase-7 appeared to activate mutually. Furthermore, we observed a significant decrease in mitochondrial membrane potential (flow cytometric analysis) and cytochrome c release (confocal microscopy, western blot after cell fractionation) from mitochondria in SK-BR-3 cells. No such changes were observed in MCF-7 cells after taxane treatment. CONCLUSION: We conclude that the activation of apical caspase-2 results in the activation of caspase-3 and -7 without the involvement of mitochondria. Caspase-9 can be activated directly via caspase-2 or alternatively after cytochrome c release from mitochondria. Subsequently, caspase-9 activation can also lead to caspase-3 and -7 activations. Caspase-3 and caspase-7 activate mutually. It seems that there is also a parallel pathway involving mitochondria that can cooperate in taxane-induced cell death in breast cancer cells.

Differentially expressed proteins in human MCF-7 breast cancer cells sensitive and resistant to paclitaxel.

Resistance of cancer cells to chemotherapeutic agents is one of the main causes of treatment failure. In order to detect proteins potentially involved in the mechanism of resistance to taxanes, we assessed differences in protein expression in MCF-7 breast cancer cells that are sensitive to paclitaxel and in the same cells with acquired resistance to paclitaxel (established in our lab). Proteins were separated using two-dimensional electrophoresis. Changes in their expression were determined and proteins with altered expression were identified using mass spectrometry. Changes in their expression were confirmed using western blot analysis. With these techniques, we found three proteins expressed differently in resistant MCF-7 cells, i.e., thyroid hormone-interacting protein 6 (TRIP6; upregulated to 650%), heat shock protein 27 (HSP27; downregulated to 50%) and cathepsin D (downregulated to 28%). Silencing of TRIP6 expression by specific siRNA leads to decreased number of grown resistant MCF-7 cells. In the present study we have pointed at some new directions in the studies of the mechanism of resistance to paclitaxel in breast cancer cells.

Role of duodenal iron transporters and hepcidin in patients with alcoholic liver disease.

Patients with alcoholic liver disease (ALD) often display disturbed iron indices. Hepcidin, a key regulator of iron metabolism, has been shown to be down-regulated by alcohol in cell lines and animal models. This down-regulation led to increased duodenal iron transport and absorption in animals. In this study, we investigated gene expression of duodenal iron transport molecules and hepcidin in three groups of patients with ALD (with anaemia, with iron overload and without iron overload) and controls. Expression of DMT1, FPN1, DCYTB, HEPH, HFE and TFR1 was measured in duodenal biopsies by using real-time PCR and Western blot. Serum hepcidin levels were measured by using ELISA. Serum hepcidin was decreased in patients with ALD. At the mRNA level, expressions of DMT1, FPN1 and TFR1 genes were significantly increased in ALD. This pattern was even more pronounced in the subgroups of patients without iron overload and with anaemia. Protein expression of FPN1 paralleled the increase at the mRNA level in the group of patients with ALD. Serum ferritin was negatively correlated with DMT1 mRNA. The down-regulation of hepcidin expression leading to up-regulation of iron transporters expression in the duodenum seems to explain iron metabolism disturbances in ALD. Alcohol consumption very probably causes suppression of hepcidin expression in patients with ALD.

Alcohol dehydrogenase and cytochrome P450 2E1 can be induced by long-term exposure to ethanol in cultured liver HEP-G2 cells.

It has been shown in previous studies that liver HEP-G2 cells (human hepatocellular carcinoma) lose their ability to express active alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1). Although both are ethanol-inducible enzymes, short-term exposure to ethanol does not cause any changes in expression or activity in cultured HEP-G2 cells. Therefore, we tested the effect of long-term exposure to ethanol on the expression and activity of both ADH and CYP2E1 in these cells. The expression of ADH and CYP2E1 was assessed at the mRNA and/or protein level using real-time PCR and Western blot analysis. Specific colorimetric assays were used for the measurement of ADH and CYP2E1 enzymatic activities. Caco-2 cells (active CYP2E1 and inactive ADH) were used as control cells. Significantly increased protein expression of ADH (about 2.5-fold) as well as CYP2E1 (about 1.6-fold) was found in HEP-G2 cells after long-term (12 mo) exposure to ethanol. The activity of ADH and CYP2E1 was also significantly increased from 12 ± 3 and 6 ± 1 nmol/h/mg of total protein to 191 ± 9 and 57 ± 9 nmol/h/mg of total protein, respectively. We suggest that the loss of activity of ethanol-metabolizing enzymes in cultured HEP-G2 cells is reversible and can be induced by prolonged exposure to ethanol. We are therefore able to reactivate HEP-G2 cells metabolic functions concerning ethanol oxidation just by modification of in vitro culture conditions without necessity of transfection with its side effect - enzyme overexpression.

Caspase-2 is involved in cell death induction by taxanes in breast cancer cells.

BACKGROUND: We studied the role of caspase-2 in apoptosis induction by taxanes (paclitaxel, novel taxane SB-T-1216) in breast cancer cells using SK-BR-3 (nonfunctional p53, functional caspase-3) and MCF-7 (functional p53, nonfunctional caspase-3) cell lines. RESULTS: Both taxanes induced apoptosis in SK-BR-3 as well as MCF-7 cells. Caspase-2 activity in SK-BR-3 cells increased approximately 15-fold within 48 h after the application of both taxanes at the death-inducing concentration (100 nM). In MCF-7 cells, caspase-2 activity increased approximately 11-fold within 60 h after the application of taxanes (300 nM). Caspase-2 activation was confirmed by decreasing levels of procaspase-2, increasing levels of cleaved caspase-2 and the cleavage of caspase-2 substrate golgin-160. The inhibition of caspase-2 expression using siRNA increased the number of surviving cells more than 2-fold in MCF-7 cells, and at least 4-fold in SK-BR-3 cells, 96 h after the application of death-inducing concentration of taxanes. The inhibition of caspase-2 expression also resulted in decreased cleavage of initiator caspases (caspase-8, caspase-9) as well as executioner caspases (caspase-3, caspase-7) in both cell lines after the application of taxanes. In control cells, caspase-2 seemed to be mainly localized in the nucleus. After the application of taxanes, it was released from the nucleus to the cytosol, due to the long-term disintegration of the nuclear envelope, in both cell lines. Taxane application led to some formation of PIDDosome complex in both cell lines within 24 h after the application. After taxane application, p21WAF1/CIP1 expression was only induced in MCF-7 cells with functional p53. However, taxane application did not result in a significant increase of PIDD expression in either SK-BR-3 or MCF-7 cells. The inhibition of RAIDD expression using siRNA did not affect the number of surviving SK-BR-3 and MCF-7 cells after taxane application at all. CONCLUSION: Caspase-2 is required, at least partially, for apoptosis induction by taxanes in tested breast cancer cells. We suggest that caspase-2 plays the role of an apical caspase in these cells. Caspase-2 seems to be activated via other mechanism than PIDDosome formation. It follows the release of caspase-2 from the nucleus to the cytosol.

Caspase-2 and JNK activated by saturated fatty acids are not involved in apoptosis induction but modulate ER stress in human pancreatic ß-cells.

BACKGROUND: Fatty acid-induced apoptosis and ER stress of pancreatic ß-cells contribute to the development of type 2 diabetes, however, the molecular mechanisms involved are unclear. AIMS: In this study we have tested the role of caspase-2 and suggested ER stress mediator JNK in saturated fatty acid-induced apoptosis of the human pancreatic ß-cells NES2Y. RESULTS: We found that stearic acid at apoptosis-inducing concentration activated ER stress signaling pathways, i.e. IRE1α, PERK and ATF6 pathways, in NES2Y cells. During stearic acid-induced apoptosis, JNK inhibition did not decrease the rate of apoptosis nor the activation of caspase-8, -9, -7 and -2 and PARP cleavage. In addition, inhibition of JNK activity did not affect CHOP expression although it did decrease the induction of BiP expression after stearic acid treatment. Caspase-2 silencing had no effect on PARP as well as caspase-8, -9 and -7 cleavage and the induction of CHOP expression, however, it also decreased the induction of BiP expression. Surprisingly, caspase-2 silencing was accompanied by increased phosphorylation of c-Jun. CONCLUSIONS: We have demonstrated that caspase-2 as well as JNK are not key players in apoptosis induction by saturated fatty acids in human pancreatic ß-cells NES2Y. However, they appear to be involved in the modulation of saturated fatty acid-induced ER stress signaling, probably by a mechanism independent of c-Jun phosphorylation.

Duodenal expression of iron transport molecules in patients with hereditary hemochromatosis or iron deficiency.

Disturbances of iron metabolism are observed in chronic liver diseases. In the present study, we examined gene expression of duodenal iron transport molecules and hepcidin in patients with hereditary hemochromatosis (HHC) (treated and untreated), involving various genotypes (genotypes which represent risk for HHC were examined), and in patients with iron deficiency anaemia (IDA). Gene expressions of DMT1, ferroportin, Dcytb, hephaestin, HFE and TFR1 were measured in duodenal biopsies using real-time PCR and Western blot. Serum hepcidin levels were measured using ELISA. DMT1, ferroportin and TFR1 mRNA levels were significantly increased in post-phlebotomized hemochromatics relative to controls. mRNAs of all tested molecules were significantly increased in patients with IDA compared to controls. The protein expression of ferroportin was increased in both groups of patients but not significantly. Spearman rank correlations showed that DMT1 versus ferroportin, Dcytb versus hephaestin and DMT1 versus TFR1 mRNAs were positively correlated regardless of the underlying cause, similarly to protein levels of ferroportin versus Dcytb and ferroportin versus hephaestin. Serum ferritin was negatively correlated with DMT1 mRNA in investigated groups of patients, except for HHC group. A decrease of serum hepcidin was observed in IDA patients, but this was not statistically significant. Our data showed that although untreated HHC patients do not have increased mRNA levels of iron transport molecules when compared to normal subjects, the expression is relatively increased in relation to body iron stores. On the other hand, post-phlebotomized HHC patients had increased DMT1 and ferroportin mRNA levels possibly due to stimulated erythropoiesis after phlebotomy.

Differing expression of genes involved in non-transferrin iron transport across plasma membrane in various cell types under iron deficiency and excess.

We studied the effect of iron deficiency, i.e., 24-h preincubation in iron-free medium, and the effect of high level of non-transferrin iron, i.e., the preincubation in ferric citrate medium containing 500 microM ferric citrate, on the expression of DMT1, Dcytb, ferroportin, hephaestin, and ceruloplasmin in various functional types of human cells. The expression of these proteins potentially involved in non-transferrin iron transport across cell membranes was tested on mRNA level by quantitative real-time PCR as well as on protein level by western blot analysis in Caco-2 (colorectal carcinoma), K562 (erythroleukemia), and HEP-G2 (hepatocellular carcinoma) cells. We found that changes in non-transferrin iron availability, i.e., iron deficiency and high level of non-transferrin iron, affect the expression of tested proteins in a cell type-specific manner. We also demonstrated that changes in the expression on mRNA level do not often correlate with relevant changes on protein level.