Abstract
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.
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.
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.
•Expression of all ABC transporters in paclitaxel-resistant sublines of SK-BR-3 and MCF-7 cells was analyzed.•SK-BR-3 and MCF-7 cells are unable to develop resistance to some taxanes.•Some taxanes are able to overcome developed resistance to paclitaxel.•Paclitaxel resistance was associated with increased levels of ABCB1 and ABCC3 protein.•ABCB1 silencing increased significantly sensitivity to both paclitaxel and doxorubicin.
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.
•MCF-7 cells sensitive and resistant to paclitaxel were compared.•2D-electrophoresis was employed to compare their total protein expression.•Protein levels of HSP27 and cathepsin D were decreased in resistant cells.•Protein level of TRIP6 was increased in resistant cells.•Silencing of TRIP6 led to decreased growth of resistant cells.
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.
A limited number of studies are devoted to regulating
expression in cancer. Hence, we aimed to unveil the regulation of
expression in MCF-7 breast cancer cells (with high
expression) and ...taxane-resistant MCF-7 sublines (manifesting even higher
expression). We found that
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,
co-amplification with the neighboring
gene, as witnessed by fluorescence in situ hybridization (FISH), led to
overexpression. Ultimately, we found high
mRNA levels in progesterone receptor-positive breast cancer and samples resected from premenopausal women.
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.
•Taxane C3′ and C3′N substituents determine whether taxane overcomes acquired resistance.•At least one non-aromatic substituent at these positions produces capability of overcoming acquired resistance.•Transport of taxanes with such substituent out of cancer cells seems to be less effective.
Saturated stearic acid (SA) induces apoptosis in the human pancreatic β-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 β-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.
Background: Fatty acid-induced apoptosis and ER stress of pancreatic β-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 β-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 β-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.
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 ...β-cell death induction by saturated fatty acids and its inhibition by unsaturated fatty acids.
Employing 2DE analysis and subsequent western blot confirmation, the differences in membrane/membrane-associated protein expression in human β-cell line NES2Y are assessed during cell death induction by stearate and its inhibition by oleate.
Induction of apoptosis by stearate is associated with significantly increased levels of Hsp90β, 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.
Several membrane-associated proteins that could be related to pro- and anti-apoptotic signaling initiated by fatty acids in human pancreatic β-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 β-cell viability.
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