Galactosylceramide Upregulates the Expression of the BCL2 Gene and Downregulates the Expression of TNFRSF1B and TNFRSF9 Genes, Acting as an Anti-Apoptotic Molecule in Breast Cancer Cells.

Galactosylceramide (GalCer) increases the resistance of breast cancer cells to doxorubicin, paclitaxel, and cisplatin by acting as an anti-apoptotic molecule. GalCer was found to specifically downregulate the levels of the pro-apoptotic TNFRSF1B and TNFRSF9 genes and upregulate the levels of the anti-apoptotic BCL2 gene, suggesting that this glycosphingolipid regulates their expression at the transcriptional level. Consistent with this hypothesis, MDA-MB-231 and MCF7 breast cancer cells with high levels of GalCer showed lower activity of the TNFRSF1B and TNFRSF9 promoters than cells lacking GalCer. In contrast, the activity of the BCL2 promoter was higher in MCF7 cells overproducing GalCer than in MCF7 cells without GalCer. However, no difference in BCL2 promoter activity was observed between MDA-MB-231 cells with high and no GalCer content. Instead, we found that high levels of GalCer increased the stability of Bcl-2 mRNA. Subsequent studies showed that breast cancer cells with high levels of GalCer are characterized by significantly lower expression of P53. Importantly, inhibition of P53 expression by siRNA in MCF7 and MDA-MB-231 cells lacking GalCer resulted in decreased expression and promoter activity of the TNFRS1B and TNFRSF9 genes. On the other hand, increased expression and promoter activity of the BCL2 gene was found in such MCF7 cells, and increased stability of Bcl-2 transcripts was observed in such MDA-MB-231 cells. Taken together, these data strongly suggest that the regulatory protein that simultaneously increases the expression of the TNFRSF1B and TNFRSF9 genes and decreases the expression of the BCL2 gene and the stability of Bcl-2 transcripts is most likely P53, the expression of which is GalCer dependent.

Prolactin-induced protein (PIP) increases the sensitivity of breast cancer cells to drug-induced apoptosis.

We have previously shown that high expression of prolactin-induced protein (PIP) correlates with the response of breast cancer (BC) patients to standard adjuvant chemotherapy (doxorubicin and cyclophosphamide), which suggests that the absence of this glycoprotein is associated with resistance of tumor cells to chemotherapy. Therefore, in the present study, we analyzed the impact of PIP expression on resistance of BC cells to anti-cancer drugs and its biological role in BC progression. Expression of PIP and apoptotic genes in BC cell lines was analyzed using real-time PCR and Western blotting. PIP was detected in BC tissue specimens using immunohistochemistry. The tumorigenicity of cancer cells was analyzed by the in vivo tumor growth assay. Apoptotic cells were detected based on caspase-3 activation, Annexin V binding and TUNEL assay. The interaction of PIP with BC cells was analyzed using flow cytometry. Using two cellular models of BC (i.e. T47D cells with the knockdown of the PIP gene and MDA-MB-231 cells overexpressing PIP), we found that high expression of PIP resulted in (1) increased sensitivity of BC cells to apoptosis induced by doxorubicin (DOX), 4-hydroperoxycyclophosphamide (4-HC), and paclitaxel (PAX), and (2) improved efficacy of anti-cancer therapy with DOX in the xenograft mice model. Accordingly, a clinical study revealed that BC patients with higher PIP expression were characterized by longer 5-year overall survival and disease-free survival. Subsequent studies showed that PIP up-regulated the expression of the following pro-apoptotic genes: CRADD, DAPK1, FASLG, CD40 and BNIP2. This pro-apoptotic activity is mediated by secreted PIP and most probably involves the specific surface receptor. This study demonstrates that a high expression level of PIP sensitizes BC cells to anti-cancer drugs. Increased sensitivity to chemotherapy is the result of pro-apoptotic activity of PIP, which is evidenced by up-regulation of specific pro-apoptotic genes. As high expression of PIP significantly correlated with a better response of patients to anti-cancer drugs, this glycoprotein can be a marker for the prognostic evaluation of adjuvant chemotherapy.

Membrane properties modulation by SanA: implications for xenobiotic resistance in Salmonella Typhimurium.

Introduction: Multidrug resistance in bacteria is a pressing concern, particularly among clinical isolates. Gram-negative bacteria like Salmonella employ various strategies, such as altering membrane properties, to resist treatment. Their two-membrane structure affects susceptibility to antibiotics, whereas specific proteins and the peptidoglycan layer maintain envelope integrity. Disruptions can compromise stability and resistance profile toward xenobiotics. In this study, we investigated the unexplored protein SanA's role in modifying bacterial membranes, impacting antibiotic resistance, and intracellular replication within host cells. Methods: We generated a sanA deletion mutant and complemented it in trans to assess its biological function. High-throughput phenotypic profiling with Biolog Phenotype microarrays was conducted using 240 xenobiotics. Membrane properties and permeability were analyzed via cytochrome c binding, hexadecane adhesion, nile red, and ethidium bromide uptake assays, respectively. For intracellular replication analysis, primary bone marrow macrophages served as a host cells model. Results: Our findings demonstrated that the absence of sanA increased membrane permeability, hydrophilicity, and positive charge, resulting in enhanced resistance to certain antibiotics that target peptidoglycan synthesis. Furthermore, the sanA deletion mutant demonstrated enhanced replication rates within primary macrophages, highlighting its ability to evade the bactericidal effects of the immune system. Taking together, we provide valuable insights into a poorly known SanA protein, highlighting the complex interplay among bacterial genetics, membrane physiology, and antibiotic resistance, underscoring its significance in understanding Salmonella pathogenicity.

Grade-dependent changes in sphingolipid metabolism in clear cell renal cell carcinoma.

There is a host of evidence for the role of bioactive sphingolipids in cancer biology, and dysregulated sphingolipid metabolism was observed in many malignant tumors. The aim of the present study was to provide more detailed data on sphingolipid metabolism in different stages of clear cell renal cell carcinoma (ccRCC). Samples of the tumor and noncancerous fragments of the same kidney were collected from patients who underwent a radical nephrectomy. The subjects were stratified according to the degree of malignancy of the tumor (n = 14 for G2, 12 for G3, and 9 for G4). The content of bioactive sphingolipids/glycosphingolipids was measured with an HPLC and HPTLC method, and the mRNA and protein expression of sphingolipid transporters and metabolizing enzymes was evaluated using real-time polymerase chain reaction (PCR) and Western blot, respectively. Compared to healthy kidney tissue, ccRCC was characterized by accumulation of sphingosine, sphingosine-1-phosphate (S1P), ceramide, dihydrosphingosine, and dihydroceramide. However, in the case of the latter two, the accumulation was limited to higher malignancy grades. In addition, compared to the healthy tissue, the content of gangliosides in the tumor was increased at the expense of globosides. We also found profound grade-dependent changes in the mRNA level of S1P-metabolizing enzymes, and spinster homolog 2. In general, their expression was much higher in G2 tumors compared to higher malignancy grades. We conclude that ccRCC is characterized by profound and multilevel alterations in sphingolipid metabolism, which to a large extent are grade-dependent. We hypothesize that dysregulation of sphingolipid metabolism contributes to the progression of ccRCC.

Glucosylceramide and galactosylceramide, small glycosphingolipids with significant impact on health and disease.

Numerous clinical observations and exploitation of cellular and animal models indicate that glucosylceramide (GlcCer) and galactosylceramide (GalCer) are involved in many physiological and pathological phenomena. In many cases, the biological importance of these monohexosylcermides has been shown indirectly as the result of studies on enzymes involved in their synthesis and degradation. Under physiological conditions, GalCer plays a key role in the maintenance of proper structure and stability of myelin and differentiation of oligodendrocytes. On the other hand, GlcCer is necessary for the proper functions of epidermis. Such an important lysosomal storage disease as Gaucher disease (GD) and a neurodegenerative disorder as Parkinson's disease are characterized by mutations in the GBA1 gene, decreased activity of lysosomal GBA1 glucosylceramidase and accumulation of GlcCer. In contrast, another lysosomal disease, Krabbe disease, is associated with mutations in the GALC gene, resulting in deficiency or decreased activity of lysosomal galactosylceramidase and accumulation of GalCer and galactosylsphingosine. Little is known about the role of both monohexosylceramides in tumor progression; however, numerous studies indicate that GlcCer and GalCer play important roles in the development of multidrug-resistance by cancer cells. It was shown that GlcCer is able to provoke immune reaction and acts as a self-antigen in GD. On the other hand, GalCer was recognized as an important cellular receptor for HIV-1. Altogether, these two molecules are excellent examples of how slight differences in chemical composition and molecular conformation contribute to profound differences in their physicochemical properties and biological functions.

Sulfatide decreases the resistance to stress-induced apoptosis and increases P-selectin-mediated adhesion: a two-edged sword in breast cancer progression.

BACKGROUND: We have previously shown that galactosylceramide (GalCer) affects the tumourigenic and metastatic properties of breast cancer cells by acting as an anti-apoptotic molecule. Since GalCer is a precursor molecule in the synthesis of sulfatides, the present study was aimed to define the role of sulfatides in apoptosis and breast cancer progression. METHODS: Expression of GAL3ST1 in breast cancer cell lines and breast cancer tissue specimens was analysed using real-time PCR, western blotting and immunohistochemistry analysis. The amount of sulfatide, GalCer and ceramide was analysed by thin-layer chromatography binding assay and by the modified hydrophilic interaction liquid chromatography coupled with electrospray mass spectrometry methodology. The tumourigenicity of cancer cells was analysed by an in-vivo tumour growth assay. Apoptotic cells were detected based on caspase-3 activation and the TUNEL assay. The interaction of breast cancer cells with P-selectin or E-selectin was analysed using the flow adhesion assay. The ability of sulfatide-expressing cells to activate and aggregate platelets was studied using the flow-cytometry-based aggregation assay. RESULTS: Using two models of breast cancer, T47D cells with blocked synthesis of sulfatide and MDA-MB-231 cells with neosynthesis of this glycosphingolipid, we showed that high sulfatide levels resulted in increased sensitivity of cancer cells to apoptosis induced by hypoxia and doxorubicin in vitro, and decreased their tumourigenicity after transplantation into athymic nu/nu mice. Accordingly, a clinical study on GAL3ST1 expression in invasive ductal carcinoma revealed that its elevated level is associated with better prognosis. Using MDA-MB-231 cells with neosynthesis of sulfatide we also showed that sulfatide is responsible for adhesion of breast cancer cells to P-selectin-expressing cells, including platelets. Sulfatide also acted as an activating molecule, increasing the expression of P-selectin. CONCLUSIONS: This study demonstrates that increased synthesis of sulfatide sensitises cancer cells to microenvironmental stress factors such as hypoxia and anticancer drugs such as doxorubicin. However, sulfatide is probably not directly involved in apoptotic cascades, because its increased synthesis by GAL3ST1 decreased the amounts of its precursor, GalCer, a known anti-apoptotic molecule. On the other hand, our data support the view that sulfatides are malignancy-related adhesive molecules involved in activating and binding P-selectin-expressing platelets to breast cancer cells.

Mucosal Autoimmunity to Cell-Bound GP2 Isoforms Is a Sensitive Marker in PSC and Associated With the Clinical Phenotype.

Introduction: Zymogen granule glycoprotein 2 (GP2) was demonstrated as first autoimmune mucosal target in primary sclerosing cholangitis (PSC) associated with disease severity. Autoantibodies to four GP2 isoforms (aGP21-4) were found in patients with inflammatory bowel diseases but reactivity against specific GP2 epitopes has not been investigated in PSC yet. Hence, the prevalence of aGP21-4 and their association with the PSC phenotype for risk prediction were examined. Methods: GP2 isoforms were stably expressed as glycosylphosphatidyl - inositol-anchored molecules in the membrane of HEp-2 cells and used as autoantigenic targets in indirect immunofluorescence assay (IFA). aGP21-4 IgA and IgG were detected by IFA in 212 PSC patients of four European university hospitals and 145 controls comprising 95 patients with cystic fibrosis and 50 healthy subjects. Results: Combined aGP21 and aGP24 IgA testing with a sensitivity of 66.0% and a specificity of 97.9% resulted in the best diagnostic performance (Youden index: 0.64) regarding all aGP2 and combinations thereof. aGP24 IgA positivity is significantly associated with the presence of cirrhosis in PSC (p = 0.0056). Logistic regression revealed the occurrence of aGP21 IgA (odds ratio [OR] 1.38, 95% confidence interval [CI]: 1.03-1.86) and aGP24 IgA (OR 1.52, 95%CI: 1.07-2.15) along with male gender (OR 0.51, 95%CI: 0.27-0.97) and older age (OR 1.03 95%CI: 1.01-1.05) as significant risks for the concomitant presence of cirrhosis in PSC. Conclusions: Combined aGP21 and aGP24 IgA analysis is preferred to single aGP2 isoform analysis for sensitive PSC autoantibody testing. Positivity for aGP21 and aGP24 IgA is associated with cirrhosis in PSC and could be used for risk stratification.

Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.

In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such podoplanin-mediated effects can affect tube formation by endothelial cells and participate in their pathological properties in the tumor context. Our experimental data were supported by clinical studies. First, when IDC and DCIS were analyzed by immunohistochemistry according to the presence of podoplanin-expressing cells, the numbers of cancer-associated fibroblasts with high expression of this glycoprotein were significantly higher in IDC than in DCIS cases. Second, using immunofluorescence, the co-localization of PDPN-positive CAFs with blood vessels stained with antibody directed against CD34 was observed in tumor stroma of IDC samples.

The Novel Type 1 Fimbriae FimH Receptor Calreticulin Plays a Role in Salmonella Host Specificity.

It was suggested that minor differences in the structure of FimH are most likely associated with differences in its adhesion specificities and may determine the tropism of various Salmonella serovars to different species and tissues. We have recently shown that FimH adhesins from host-adapted serovars, e.g., Salmonella Choleraesuis (SCh), bind to other glycoprotein receptors compared to FimH from host-unrestricted Salmonella Enteritidis (SE). Here we identify porcine calreticulin expressed by swine intestinal cells as a host-specific receptor for SCh FimH adhesin, suggesting that such an interaction may contribute to SCh host specificity. Calreticulin was identified by 2D electrophoresis and mass spectrometry as a glycoprotein that was bound specifically by recombinant SCh FimH protein, but not by FimH from SE. The functionality of calreticulin as a specific receptor of SCh FimH adhesin was further confirmed by adhesion and invasion of mutated strains of SCh carrying different variants of FimH proteins to IPEC-J2 cells with overexpression and silenced expression of calreticulin. It was found that SCh carrying the active variant of FimH adhered and invaded IPEC-J2 cells with calreticulin overexpression at significantly higher numbers than those of SCh expressing the non-active variant or SE variant of FimH. Moreover, binding of SCh carrying the active variant of FimH to IPEC-J2 with silenced calreticulin expression was significantly weaker. Furthermore, we observed that SCh infection induces translocation of calreticulin to cell membrane. All of the aforementioned results lead to the general conclusion that Salmonella host specificity requires not only special mechanisms and proteins expressed by the pathogen but also specifically recognized receptors expressed by a specific host.

Podoplanin - a small glycoprotein with many faces.

Podoplanin is a small membrane glycoprotein with a large number of O-glycoside chains and therefore it belongs to mucin-type proteins. It can be found on the surface of many types of normal cells originating from various germ layers. It is present primarily on the endothelium of lymphatic vessels, type I pneumocytes and glomerular podocytes. Increased levels of podoplanin or its neo-expression have been found in numerous types of human carcinomas, but it is especially common in squamous cell carcinomas, such as cervical, larynx, oral cavity, skin and lung cancer. This small sialomucin is also seen on the surface of cancer-associated fibroblasts (CAFs) in lung adenocarcinomas, as well as in breast and pancreatic tumors. In most cancers, a high level of podoplanin expression, both in cancer cells, as well as in CAFs, is correlated with an increased incidence of metastasis to lymph nodes and shorter survival time of patients. Little is known about the biological role of podoplanin, however research carried out on mice with a knock-out gene of this glycoprotein shows that the presence of podoplanin determines normal development of lungs, the lymphatic system and heart. Podoplanin on cancer cells and CAFs seems to play an important role in the development and progression of various cancers. However, its role in these processes is both unclear and controversial. In this review, the role of podoplanin in both physiological processes and carcinogenesis is discussed.

[The biological role of sulfatides]. / Sulfatydy i ich rola biologiczna.

Sulfatides (3-O-sulfogalactosylceramides, sulfated galactocerebrosides, SM4) are esters of sulfuric acid with galactosylceramides. These acidic glycosphingolipids, present at the external leaflet of the plasma membrane, are synthesized by a variety of mammalian cells. They are especially abundant in the myelin sheath of oligodendrocytes in the central nervous system and Schwann cells in the peripheral nervous system. Studies using cerebroside galactosyltransferase-deficient mice revealed that sulfatides are responsible for proper structure and functioning of myelin. Large amounts of sulfatides are also found in the kidney, gastrointestinal tract, islets of Langerhans, and membranes of erythrocytes, thrombocytes and granulocytes. They are ligands for numerous proteins, but in most cases the biological role of such interactions is poorly understood. A notable exception is their binding by P- and L-selectins. Platelet sulfatides are major ligands for P-selectin, and this interaction is critical for the formation of stable platelet aggregates. Sulfatides also bind to chemokines, and seem to play a role in regulation of cytokine expression in human lymphocytes and monocytes. Aberrant metabolism of sulfatides, could cause several important human diseases. In this article, we describe the changes in sulfatide expression associated with such nervous disorders as metachromatic leukodystrophy (MLD), Parkinson's disease and Alzheimer's disease, and several types of cancer, e.g. colon cancer, kidney cancer, and ovarian cancer. We also discuss the involvement of sulfatides in cancer progression, diabetes and autoimmune and immune disorders such as multiple sclerosis. This acidic glycosphingolipids seem to play an important role in pathogenesis of infectious diseases, serving as receptors for binding various bacteria and viruses.

Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression.

It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients' shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases.

Galactosylceramide affects tumorigenic and metastatic properties of breast cancer cells as an anti-apoptotic molecule.

It was recently proposed that UDP-galactose:ceramide galactosyltransferase (UGT8), enzyme responsible for synthesis of galactosylceramide (GalCer), is a significant index of tumor aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer. To further reveal the role of UGT8 and GalCer in breast cancer progression, tumorigenicity and metastatic potential of control MDA-MB-231 cells (MDA/LUC) and MDA-MB-231 cells (MDA/LUC-shUGT8) with highly decreased expression of UGT8 and GalCer after stable expression of shRNA directed against UGT8 mRNA was studied in vivo in athymic nu/nu mice. Control MDA/LUC cells formed tumors and metastatic colonies much more efficiently in comparison to MDA/LUC-shUGT8 cells with suppressed synthesis of GalCer after their, respectively, orthotopic and intracardiac transplantation. These findings indicate that UGT8 and GalCer have a profound effect on tumorigenic and metastatic properties of breast cancer cells. In accordance with this finding, immunohistochemical staining of tumor specimens revealed that high expression of UGT8 accompanied by accumulation of GalCer in MDA-MB-231 cells is associated with a much higher proliferative index and a lower number of apoptotic cells in comparison to the MDA/LUC-shUGT8 cells. In addition, it was found that expression of UGT8 in MDA-MB-231 cells increased their resistance to apoptosis induced by doxorubicin in vitro. Therefore, these data suggest that accumulation of GalCer in tumor cells inhibits apoptosis, which would facilitates metastatic cells to survive in the hostile microenvironment of tumor in target organ.