Semispecific TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) displays cytotoxic activity by induction of apoptosis, autophagy and protein aggregation in U937 cells

Introduction. The main component of extralysosomal proteolysis is the ubiquitin-proteasome system (UPS), which is supplemented by tripeptidyl peptidase II (TPPII). That system is a target for anticancer strategies by using proteasome inhibitors. Data from several studies on leukemic cells share evidence for the beneficial and potential role of TPPII in cell survivability. Therefore, the aim of this work was to analyze the effect of AAF-cmk, a membrane permeable semi-specific TPPII inhibitor, on human monocytic leukemic cells U937 for translational research. Material and methods. We studied the viability of U937 cells incubated with AAF-cmk using tetrazolium salt reduction assay (MTT) and apoptosis induction by assessing caspase activation by Western blotting and Annexin V binding assays. Transmission electron microscopy (TEM), a gold standard for apoptosis and autophagy detection, was used to assess the ultrastructure of U937 cells. Results. Incubation of cells with AAF-cmk reduced their viability and induced apoptosis by intrinsic pathway. In groups treated with AAF-cmk, activation of caspases 9 and 3 was observed and caspase inhibition by zVDA restored cell viability. TEM revealed the presence of ultrastructural features of apoptosis and authophagy. Moreover, we identified two types of protein aggregates. The first one was found in close proximity to the endoplasmic reticulum (ER) and corresponds to Aggresome-Like Structure (ALIS); however, the second novel type of aggregate was not related to ER elements, but rather to free cytosolic ribosomes. This type did not correspond to the aggresome neither in localization nor the structure, thus we referred these aggregates as ALiSNER (Aggresome-Like Structure Not Associated With the ER). Conclusions. Our results provide novel and important findings about the role of TPPII in protein homeostasis and cell survival. Since semispecific TPPII inhibitor AAF-cmk displays cytotoxic activity against leukemic U937 cells in vitro it can be considered as a potential anticancer agent

Protein-based identification of quantitative trait loci associated with malignant transformation in two HER2+ cellular models of breast cancer

Background A contemporary view of the cancer genome reveals extensive rearrangement compared to normal cells. Yet how these genetic alterations translate into specific proteomic changes that underpin acquiring the hallmarks of cancer remains unresolved. The objectives of this study were to quantify alterations in protein expression in two HER2+ cellular models of breast cancer and to infer differentially regulated signaling pathways in these models associated with the hallmarks of cancer. Results A proteomic workflow was used to identify proteins in two HER2 positive tumorigenic cell lines (BT474 and SKBR3) that were differentially expressed relative to a normal human mammary epithelial cell line (184A1). A total of 64 (BT474-184A1) and 69 (SKBR3-184A1) proteins were uniquely identified that were differentially expressed by at least 1.5-fold. Pathway inference tools were used to interpret these proteins in terms of functionally enriched pathways in the tumor cell lines. We observed protein ubiquitination and apoptosis signaling pathways were both enriched in the two breast cancer models while IGF signaling and cell motility pathways were enriched in BT474 and amino acid metabolism were enriched in the SKBR3 cell line. Conclusion While protein ubiquitination and apoptosis signaling pathways were common to both the cell lines, the observed patterns of protein expression suggest that the evasion of apoptosis in each tumorigenic cell line occurs via different mechanisms. Evidently, apoptosis is regulated in BT474 via down regulation of Bid and in SKBR3 via up regulation of Calpain-11 as compared to 184A1

The effect of PCB126, 77, and 153 on the intracellular mobilization of Caplus 2 in bovine granulosa and luteal cells after FSH and LH surge in vitro

Polychlorinated biphenyls (PCBs) are a group of persistent environmental pollutants that impair cattle reproduction. Among other effects, PCBs can disturb the intracellular mobilization of Ca²⁺ in several cell types. Hence, it is possible that they disrupt the transduction of intracellular signals generated from gonadotropin (FSH/LH) receptors. In steroidogenic ovarian cells, a defect in Ca²⁺ mobilization may have a detrimental influence on two important processes: the secretion of steroids (E2 or/and P4) and their morphological and functional differentiation. The aim of this study was to determine the influence of PCBs: 126 (dioxin-like) 77 (ambivalent) and 153 (estrogen-like) and a mixture of PCBs (Aroclor 1248) on these processes. Bovine granulosa and luteal cells were incubated for 72 hrs with PCBs (100 ng/ml), followed by Fura 2AM dye, and the fluctuations in intracellular Ca²⁺ mobilization after FSH/LH treatment were determined using an inverted microscope coupled with a CCD camera. The intensity and area of fluorescence excited by UV light were detected in the green spectrum of visible light. Aroclor 1248 and PCBs 153 and 77 significantly decreased (P < 0.01-0.001) the effect of FSH on intracellular Ca²⁺ mobilization in granulosa cells. In luteal cells, the most effective PCB on this process was PCB 77. The results revealed adverse effects of PCBs on the mobilization of intracellular Ca²⁺. Moreover, the estrogen-like congeners were found to more effectively disturb this process than the dioxin-like PCB 126. Hence, it is possible for PCBs to have a negative influence on reproductive processes by affecting calcium mobilization

Application of database technology to analysis of rock structure images

The aim of this paper is to present a possibility to use information technology in the form of databases for processing and analyzing large image sets based on methods of image analysis and mathematical morphology. Up to now the use of databases in the image analysis process has been reduced to storing large amounts of data in the form of images. However, all transformations and analyses of such sets are made on user’s computers. This requires a large data set (images) to be sent by network each time, and also it may possess the problems resulting from managing such large amounts of analyzed photographs on a computer. The proposed approach completely eliminates these problems by moving all transformations of image analysis to a database platform. For this purpose a set of routines realizing transformations of the image analysis and mathematical morphology was developed. The proposed approach allows the unification of the image processing and analysis area and advanced statistical analyses of obtained parameters describing geometrical sizes of objects on photographs. The proposed methodology was illustrated by practical realization of two measurement types for a simple structure of copper concentrate and more complicated, from the point of view of image analysis, structures such as dolomites from Redziny and Laskowa Gora and sandstones from Tumlin and Wisniowka

The preference of different broad bean cultivars by Sitona lineatus L. [Coleoptera, Curculionidae]

In the experiments the lifetime, survival and feeding preferences of Sitona lineatus L. on the various broad bean cultivars were investigated. The measures of the injured disks were performed by means of automatic image analysis. This method led to reduction of time required for the measures. Based on these experiments it can be suggested that Hangdown Zielony was the most preferable and suitable cultivar for the S. lineatus adults from all tested broad bean cultivars. The largest injured disk surface, the longest life and the highest survival of Sitona weevils were recorded on this variety.Celem badań było ustalenie różnic w długości życia, przeżywalności oraz preferencji pokarmowej chrząszczy Sitona lineatus L. żerujących na trzech odmianach bobu. Na podstawie przeprowadzonych obserwacji można stwierdzić, że odmiana Hangdown Zielony stanowiła najbardziej odpowiedni pokarm dla oprzędzików. Uwidoczniło się to zarówno w dużej, zjedzonej powierzchni krążków, jak i w najdłuższym okresie życia oprzędzików żerujących na tej odmianie. Pomiary dotyczące preferencji pokarmowej oprzędzika polegające na wyznaczeniu powierzchni, obwodów i współczynników kształtu wszystkich uszkodzonych liści prowadzono przy wykorzystaniu metod komputerowej analizy obrazów. Pozwoliło to na znaczną redukcję czasu niezbędnego do wykonania tych pomiarów

Sex-dependent differences in the effect of early weaning on the chosen hormones secretion in sheep during the postnatal transition to puberty – Preliminary results

The influence of early weaning on the cortisol, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and growth hormone (GH) secretion in lambs of both sexes and testosterone (T4) level in male lambs during the postnatal transition to puberty was investigated by radioimmunoassay. It was hypothesized that this influence is long-term and sexually dimorphic. Hence, the effect of weaning at 5 weeks of age in comparison with the weaning at 9 weeks of age on hormone concentrations in peripheral blood plasma of 5-, 9-, 12-, and 16-week-old lambs of both sexes was investigated. The cortisol concentrations were greater (P<0.05) in control and early weaned female lambs than in male lambs at investigated stages. Weaning at 5 weeks of age resulted in the lover (P<0.05) cortisol secretion in male lambs in contrast to the greater (P<0.05) cortisol secretion in female lambs at 16 weeks of age. Weaning at 5 weeks of age stimulated (P<0.001) the FSH secretion, but reduced (P<0.001) the LH, GH and T4 secretion in 16-week-old male lambs. In female lambs early weaning inhibited (P<0.05) the FSH secretion at 9 weeks of age, LH secretion after 9 weeks of age and GH secretion after 12 weeks of age. Thus, early weaning results in the sexually dimorphic stress reaction that is more potent and long-lasting in female in contrast to male lambs. This maternal deprivation stress contributes to the inhibition of LH and GH secretion in lambs of both sexes and T4 secretion in male lambs during the postnatal transition to puberty

Classification of Cell-in-Cell Structures: Different Phenomena with Similar Appearance

A phenomenon known for over 100 years named “cell-in-cell” (CIC) is now undergoing its renaissance, mostly due to modern cell visualization techniques. It is no longer an esoteric process studied by a few cell biologists, as there is increasing evidence that CICs may have prognostic and diagnostic value for cancer patients. There are many unresolved questions stemming from the difficulties in studying CICs and the limitations of current molecular techniques. CIC formation involves a dynamic interaction between an outer or engulfing cell and an inner or engulfed cell, which can be of the same (homotypic) or different kind (heterotypic). Either one of those cells appears to be able to initiate this process, which involves signaling through cell–cell adhesion, followed by cytoskeleton activation, leading to the deformation of the cellular membrane and movements of both cells that subsequently result in CICs. This review focuses on the distinction of five known forms of CIC (cell cannibalism, phagoptosis, enclysis, entosis, and emperipolesis), their unique features, characteristics, and underlying molecular mechanisms

Homotypic Entosis as a Potential Novel Diagnostic Marker in Breast Cancer

Homotypic entotic figures, which are a form of “cell-in-cell” structures, are considered a potential novel independent prognostic marker in various cancers. Nevertheless, the knowledge concerning the biological role of this phenomenon is still unclear. Since breast cancer cells are remarkably entosis-competent, we aimed to investigate and compare the frequency of entoses in a primary breast tumor and in its lymph node metastasis. Moreover, as there are limited data on defined molecular markers of entosis, we investigated entosis in correlation with classical breast cancer biomarkers used in routine pathomorphological diagnostics (HER2, ER, PR, and Ki67). In the study, a cohort of entosis-positive breast cancer samples paired into primary lesions and lymph node metastases was used. The inclusion criteria were a diagnosis of NOS cancer, lymph node metastases, the presence of entotic figures in the primary lesion, and/or lymph node metastases. In a selected, double-negative, HER2-positive NOS breast cancer case, entoses were characterized by a correlation between an epithelial–mesenchymal transition and proliferation markers. We observed that in the investigated cohort entotic figures were positively correlated with Ki67 and HER2, but not with ER or PR markers. Moreover, for the first time, we identified Ki67-positive mitotic inner entotic cells in clinical carcinoma samples. Our study performed on primary and secondary breast cancer specimens indicated that entotic figures, when examined by routine HE histological staining, present potential diagnostic value, since they correlate with two classical prognostic factors of breast cancer