Combination of cyclopamine and tamoxifen promotes survival and migration of MCF-7 breast cancer cells – interaction of Hedgehog-Gli and Estrogen receptor signaling pathways

Hedgehog-Gli (Hh-Gli) signaling pathway is one of the new molecular targets found upregulated in breast tumors. Estrogen receptor alpha (ERα) signaling has a key role in the development of hormone-dependent breast cancer. We aimed to investigate the effects of inhibiting both pathways simultaneously on breast cancer cell survival and the potential interactions between these two signaling pathways. ER-positive MCF-7 cells show decreased viability after treatment with cyclopamine, a Hh-Gli pathway inhibitor, as well as after tamoxifen (an ERα inhibitor) treatment. Simultaneous treatment with cyclopamine and tamoxifen on the other hand, causes short-term survival of cells, and increased migration. We found upregulated Hh-Gli signaling under these conditions and protein profiling revealed increased expression of proteins involved in cell proliferation and migration. Therefore, even though Hh-Gli signaling seems to be a good potential target for breast cancer therapy, caution must be advised, especially when combining therapies. In addition, we also show a potential direct interaction between the Shh protein and ERα in MCF-7 cells. Our data suggest that the Shh protein is able to activate ERα independently of the canonical Hh-Gli signaling pathway. Therefore, this may present an additional boost for ER-positive cells that express Shh, even in the absence of estroge

Does Body Mass Index and Position of Impacted Lower Third Molar Affect the Postoperative Pain Intensity?

The main objective of this study was to determine to which extent body mass index and position of impacted lower third molar was affecting the pain intensity in the first seven postoperative days. The study was conducted following the extraction of the lower third molar in 108 patients. Depending on the type of information given to each particular patient, the patients were divided in two groups: the test group where patients were given detailed standard written and verbal instructions and the control group which received only standard written instructions about treatment after surgery. Using canonical discriminant analysis we investigated the influence of body mass index and the position of impacted lower third molar on postoperative pain intensity in two groups of patients. Results of this study showed that the body mass index or the tooth position did not have influence on intensity of postoperative pain. The body mass index and the position of impacted lower third molar do not affect the postoperative pain intensity

Oral Health of Children from the SOS Children’s Village in Croatia

The aim of this study was to determine the values of DMFT/DMFS and dft/dfs in the examined groups of children and the assessment of the mothers of the examined groups of children related to the oral health of their children. The research included children from the SOS Children’s Village in Croatia as well as children from biological families from rural and urban areas. The children were examined by the visual–tactile method according to the standardized World Health Organization criteria. dft/DMFT and dfs/DMFS indices were calculated. An analysis of completed questionnaires was made. The children from the SOS Children’s Village demonstrated the lowest mean values of the dft/dfs (2.42/3.31) and DMFT/DMFS (1.61/2.23) indices compared to children from rural and urban areas. The Kruskal–Wallis test showed a significant difference (p = 0.01) in SiC index values between the examined children. In the groups of children from the SOS Children’s Village and from the rural area compared to the children from the urban area, oral hygiene was singled out as the most important factor in the analysis of the main components. An equally significant factor for all the respondents is the assessment of oral health and eating habits. The least significant factor for the group of children from the SOS Children’s Village is socio-economic status, which is the most significant for the children from the urban area. The children from the SOS Children’s village have the lowest dft/DMFT, dfs/DMFS, and SiC indices. The most important factor influencing oral health in the group of children from the SOS Children’s Village that stands out is oral hygiene, and the least important is the socio-economic status. The assessment of oral health by the SOS mothers does not differ from the assessment of biological mothers of children from rural and urban areas

Knowledge on Pre-Hospital Emergency Management of Tooth Avulsion among Croatian Students of the Faculty of Education

The purpose of this study was to evaluate the knowledge regarding tooth avulsion and dental first aid response among Croatian students of the Faculty of Education. A cross-sectional study was conducted among students. Participants (N = 235) were female with an average age of 21.9 ± 2.7 years. The questionnaire contained 10 close-ended questions with two to eleven possible answers. Every participant chose one correct answer. Statistical significance was determined using a Chi-square test. Majority of participants had never received any kind of information on management of a knocked-out tooth. Questions in the nature of tooth injuries, a knocked-out tooth, tooth replantation and whether the knocked-out tooth should be placed back were answered confirmatively by 40.43%, 83.40%, 57.02% and 62.55% of participants, respectively. The questionnaire showed that 18.78% of participants were aware that replantation should be performed within 30 min. The appropriate cleaning and transport medium was chosen by 40.85% and 35.31% of participants, respectively. A nearby dentist would be visited by 66.38% of students. Only 8.08% of participants provided a correct answer to all of the knowledge-based questions. The “I do not know” answer was chosen by 18.30% of participants whenever it was offered. This indicates that the majority (89.78%) were not aware of the fact that the procedure they chose would be inappropriate. The current study confirmed that future primary school teachers have a lack of knowledge for immediate response to tooth avulsion, leaving small chances for a successful prognosis of tooth replantation

Does Body Mass Index and Position of Impacted Lower Third Molar Affect the Postoperative Pain Intensity?

The main objective of this study was to determine to which extent body mass index and position of impacted lower third molar was affecting the pain intensity in the first seven postoperative days. The study was conducted following the extraction of the lower third molar in 108 patients. Depending on the type of information given to each particular patient, the patients were divided in two groups: the test group where patients were given detailed standard written and verbal instructions and the control group which received only standard written instructions about treatment after surgery. Using canonical discriminant analysis we investigated the influence of body mass index and the position of impacted lower third molar on postoperative pain intensity in two groups of patients. Results of this study showed that the body mass index or the tooth position did not have influence on intensity of postoperative pain. The body mass index and the position of impacted lower third molar do not affect the postoperative pain intensity

Effects of cyclopamine (A) and tamoxifen (B) on Hh-Gli pathway gene expression in MCF-7 and SkBr-3 cells.

<p>The Hh-Gli pathway is upregulated after short-term combined treatment in MCF-7, but the effect is negated after longer treatment. On the Western blot image, band quantification relative to actin and non-treated cells is denoted below the bands. (C). The effect of combined treatment on SkBr-3 cell line is weak (D). Gene expression levels are shown on graph as relative fold change relative to non-treated conditions with reference value 1 pointed out with emboldened bar. Only combined cyclopamine and tamoxifen treatment induces migration in MCF-7 cells. Representative images of the wound healing assay at 0 and 26 h (after processing with TScratch software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114510#pone.0114510-Gebck1" target="_blank">[23]</a>) are shown for non-treated conditions (NT; N = 16), cyclopamine treatment (CYC; N = 16), tamoxifen treatment (TAM; N = 14) and combined treatment with cyclopamine and tamoxifen (C+T; N = 12) (E). Quantitative analysis of the percentage of open wound areas is shown on the graph, (*) P<0.05 (F). Transwell migration assay confirmed increased migration capacity of cells after combined cyclopamine and tamoxifen treatment. Representative images of migrated cells after 48 h are shown for non-treated conditions (NT; N = 15), cyclopamine treatment (CYC; N = 15), tamoxifen treatment (TAM; N = 15) and combined treatment (C+T; N = 15) (G). Quantitative analysis of the relative number of migrated cells (analyzed relative to non-treated cells) is shown on graph, (*) P<0.0001 (H).</p

Differentially expressed proteins in MCF-7 cells treated with cyclopamine and tamoxifen compared with non-treated control cells.

<p>General Functions are obtained from the UniProt and NCBI Gene databases. Protein numbers correspond to the numbers marked on the 2-D gels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114510#pone.0114510.s001" target="_blank">Figure S1</a>). Numbers in the table correspond to spot numbers denoted on the 2-D gel images; missing numbers in the table are unidentified proteins or proteins with score less than 39.</p><p>Differentially expressed proteins in MCF-7 cells treated with cyclopamine and tamoxifen compared with non-treated control cells.</p

Gene and protein expression levels after transfection with <i>GLI1</i> (GLI1) and additional stimulation with Shh protein (GLI1+SHH).

<p><i>ERα</i> gene expression increases in MCF-7 cells only after additional Shh stimulation (A) while <i>ERα</i> gene expression does not change in SkBr-3 cells (B). Gli1, Ptch1 and ERα protein levels in MCF-7 cells after <i>GLI1</i> transfection and additional Shh stimulation (C). Protein bands were quantified and normalized relative to actin and non-treated conditions and the relative values are denoted below each band. Relative gene expression of <i>PTCH1</i> (D, E) and <i>ERα</i> (F,G) after silencing of <i>PTCH1</i> gene in MCF-7 and SkBr-3 cell line. Efficient silencing (<30% of residual expression) was achieved 24 h post-transfection in MCF-7 cell line, and 48 h post-transfection in SkBr-3 cell line.</p

Effect of stimulation with Shh protein on pathway activity in MCF-7 (A,C) and SkBr-3 cells (B).

<p>Gene expression levels are shown on graph as relative fold change relative to non-treated conditions with reference value 1 pointed out with emboldened bar. Relative gene expression of <i>ERα</i> after treatment with Shh protein (D,E). Non-treated cells (NT) have a relative value 1. ERα protein expression in MCF-7 cells increases after treatment with Shh protein for 48 h (F) Protein bands were quantified and normalized relative to actin and non-treated conditions and the relative values are denoted below each band.</p

Cell viability after tamoxifen (A,B), cyclopamine (C,D) or combined treatment (E,F) in MCF-7 and SkBr-3 cell lines.

<p>Tamoxifen and cyclopamine each inhibit proliferation of MCF-7 cells in a dose dependent manner (A,C). When administered simultaneously, they cause a short term survival effect in MCF-7 cells (C+T 48 h) – pointed out with arrow, whereas long term simultaneous treatment induces strong cell death in these cells (C+T 96 h). Combination treatment of cyclopamine for 48 h followed by tamoxifen for 48 h (C 48 h →T 48 h) or vice versa (T 48 h →C 48 h) showed an effect similar to tamoxifen alone (E). Tamoxifen and cyclopamine show only a mild inhibitory effect on SkBr-3 cell proliferation at longest exposures (B,D) while combined treatment has no pronounced effect (F).</p