Rostyslav Stoika

Expression of Smad proteins in human colorectal cancer

Escape from transforming growth factor‐β (TGF‐β)–induced inhibition of proliferation has been observed in many tumor cells and may contribute to loss of growth control. Smad proteins have been identified as major components in the intracellular signaling of TGF‐β family members. In this study, we examined the expression of receptor‐activated, common‐mediator and inhibitory Smads by immunohistochemistry in human colorectal cancers. We found increased expression of receptor‐activated Smads in a fraction of the tumor cells, while no immunostaining for Smad2, Smad3 or Smad5 and only occasional staining for Smad1/8 was found in epithelial mucosa of normal colon. No or only weak staining for receptor‐activated Smads, common‐mediator Smad4 and inhibitory Smads was observed in the tumor stroma. Common‐mediator Smad4 and inhibitory Smads were detected in cells of both tumor and normal tissues. We observed a distinct pattern of Smad4 immunostaining of epithelial cells along colon crypts, with high expression in zones of terminal differentiation. Our data show selective up‐regulation of receptor‐activated Smad proteins in human colorectal cancers and suggest involvement of Smad4 in differentiation and apoptosis of surface epithelial cells of normal crypts. Int. J. Cancer 82:197–202, 1999.

Macrophages Discriminate Glycosylation Patterns of Apoptotic Cell-derived Microparticles

Background: Apoptotic cells release vesicles, which expose “eat-me” signals. Results: Vesicles originated from endoplasmic reticulum expose immature glycoepitopes and are preferentially phagocytosed by macrophages. Conclusion: Immature surface glycoepitopes serve as “eat-me” signals for the clearance of apoptotic vesicles originated from endoplasmic reticulum. Significance: Understanding the distinction by macrophages of apoptotic blebs may provide new insights into clearance-related diseases. Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

Correlation of the cytotoxic activity of four different alkaloids, from Chelidonium majus (greater celandine), with their DNA intercalating properties and ability to induce breaks in the DNA of NK/Ly murine lymphoma cells

The purpose of this study was to examine the relationship between the DNA intercalating characteristics and the DNA damaging capacity of four alkaloids extracted from Chelidonium majus L, as well as their toxicity towards murine NK/Ly lymphoma cells. Chelerythrine, sanguinarine and coptisine were found to be intercalated into the DNA isolated from NK/Ly cells, meanwhile, chelidonine exhibited no affinity to DNA. Sanguinarine exhibited the greatest toxicity toward NK/Ly cells, and the toxicity of the other three decreased in descending order: chelerythrine, coptisine and chelidonine. Chelerythrine and sanguinarine caused DNA damage, illustrated by the formation of comets of the third class. Coptisine was less toxic than chelerythrine and sanguinarine, and affected the formation the same class of comets in higher concentration. The quantity of comets induced by chelidonine were negligible, a finding consistent with its inability to intercalate into DNA structure. The ability of four main alkaloids of Chelidonium majus L., to intercalate into DNA isolated from murine NK/Ly lymphoma cells, correlated with their ability to induce breaks in cellular DNA and with their toxic effect towards those cells.

Search for novel cell surface markers of apoptotic cells

Surface markers of apoptotic cells are of great interest as potential targets for non-destructive detection and study of these cells. They are also important for apoptotic cell recognition and subsequent clearance by cells of the immune system. Recently, it was found that apoptosis is accompanied by not only the loss of plasma membrane asymmetry detected by Annexin V, but also by changes in cell surface glycoconjugates. These novel markers of apoptosis are α-d-mannose and β-d-galactose-specific plasma membrane glycoproteins whose expression is substantially increased after induction of apoptosis. The glyconeoepitopes described in this article are proposed to be useful for both, the detection of apoptotic cells and the isolation of the latter, from mixed populations.

Cytochemical study of role of α-d-mannose- and β-d-galactose-containing glycoproteins in apoptosis

Recently, we found increased levels of α-d-mannose- and β-d-galactose-containing glycoproteins in plasma membrane of the apoptotic murine leukemia L1210 cells (Bilyy & Stoika 2003). That indicator was suggested to be a novel marker of apoptosis in L1210 cells. The aim of our present work was to reveal if these changes in glycoprotein expression can be common for apoptotic cells of different origin and for various ways of apoptosis induction. It was demonstrated that an elevated expression of plasma membrane glycoproteins rich in α-d-mannose and β-d-galactose did not depend on type of cell line and its tissue origin as well as on nature of apoptosis-inducing agent. We also found that an increase in membrane glycoprotein expression was dependent on concentration of apoptosis-inducing agent and was time-dependent. Changes in glycoproteins’ expression were detected as early as 9–12 hours after apoptosis induction. Two hours pretreatment of cells with non-labeled lectin decreased plasma membrane staining with corresponding peroxidase-labeled lectin, probably because of lectin-induced internalization of specific membrane glycoproteins. PSL-lectin-affinity procedure was developed for isolation of apoptotic cells from their mixed population with normal cells. Lectin-dependent agglutination analysis showed that this process occurs at much lower lectin dilutions in the apoptotic cells than in the non-apoptotic cells. Thus, we found that α-d-mannose- and β-d-galactose-containing glycoproteins can be used for lectinocytochemical detection, study and isolation of apoptotic cells.

Doxorubicin inhibits TGF-β signaling in human lung carcinoma A549 cells

Doxorubicin is a DNA-damaging drug, commonly used for treatment of cancer patients. Doxorubicin causes not only cytotoxic and cytostatic effects, but also inhibits metastasis formation, while TGFbeta1 (Transforming Growth Factor-beta1) is a cytokine that is often up-regulated in human cancers and can promote metastasis formation. We have studied the influence of Doxorubicin on TGFbeta signaling in tumor cells. Here we have demonstrated that Doxorubicin inhibited TGFbeta-signaling in human lung adenocarcinoma A549 cells, namely, it blocked TGFbeta1-induced activation of Smad3-responsive CAGA(12)-Luc reporter, but did not affect c-myc-Luc reporter. That effect was observed as early as after 1-3 h of treating these cells with Doxorubicin, while the other drugs cisplatin or methotrexate did not alter activation of CAGA(12)-Luc reporter under the same conditions. Besides, after 1 h action, Doxorubicin abrogated TGFbeta-induced translocation of Smad3-protein from the cytoplasm to the nucleus. Down-regulation of expression of Smad2, Smad3, and Smad4 proteins, and up-regulation of inhibitory Smad7 protein upon Doxorubicin treatment, were found after 12-24 h of Doxorubicin treatment. Phosphorylation of Smad2/3 proteins was also affected by Doxorubicin. Summarizing, we have found that human tumor cells treatment with Doxorubicin resulted in the inhibition of TGFbeta-signaling at both early (1 h) and later (12 h) stages of the drug action. Such inhibition can be a new potential mechanism for Doxorubicin action towards tumor cells.

In vivo expression and characteristics of novel α-d-mannose-rich glycoprotein markers of apoptotic cells

We recently established that an increased expression of α‐d‐mannose (Man)‐ and β‐d‐galactose‐rich plasma membrane glycoproteins (GPs) is characteristic for apoptotic cells in vitro [Bilyy, R.O., Stoika, R.S., 2003. Lectinocytochemical detection of apoptotic murine leukemia L1210 cells. Cytometry 56A, 89–95]. It was independent of cell line or apoptosis‐inducing agent, and can therefore be considered as a selective marker for identification and isolation of apoptotic cells [Bilyy, R.O., Antonyuk, V.O., Stoika, R.S., 2004. Cytochemical study of role of alpha‐d‐mannose‐ and beta‐d‐galactose‐containing glycoproteins in apoptosis. J. Mol. Histol. 35, 829–838]. The main goals of the present study were: (1) to determine whether an increased expression of specific GPs also takes place after apoptosis induction in vivo; and (2) to identify additional characteristics of the membrane GP markers of the apoptotic cells. To reach these goals, we studied the expression of α‐Man‐rich membrane GPs in murine leukemia L1210 cells inoculated into abdominal cavities of mice which were then subjected to the action of apoptosis inducer doxorubicin. Another experimental model used in the present work was splenocytes obtained from mice treated with dexamethasone. Lectin‐affinity chromatography and PAGE electrophoresis, or PAGE electrophoresis and lectinoblot analysis were applied for isolation of plasma membrane GPs (34 kDa, and high MW of ∼600 and 800 kDa) whose expressions were increased during apoptosis. Triton X‐114 treatment of cell membrane samples showed that the apoptotic cell‐specific GPs were localized in the peripheral and integral compartments of plasma membrane. Apoptosis in vitro and in vivo was accompanied by an increased expression of the same GP, identified by MALDI‐TOF MS analysis as the microtubule‐actin cross‐linking factor 1. Other GPs, whose expressions were also increased at apoptosis, were similarly identified as G‐protein β‐subunit like (Acc# BAA06185.1) and dystonin isoform β.

Transforming growth factor beta-1 enhances cytotoxic effect of doxorubicin in human lung adenocarcinoma cells of A549 line

Transforming growth factor beta‐1 (TGFbeta‐1) is a regulator of cell proliferation, differentiation and apoptosis. Doxorubicin (adriamycin), an anthracycline drug causing double‐strand DNA breaks, is widely used in anticancer chemotherapy. Here we demonstrated that TGFbeta‐1 enhanced cytotoxic (proapoptotic) action of doxorubicin towards cultured human lung carcinoma A549 cells. Western‐blot analysis and immunocytochemistry were used to show that doxorubicin induced PARP degradation in A549 cells, and TGFbeta‐1 enhanced that action of the drug. The obtained results suggest a possibility of biomodulating effect of TGFbeta‐1 on tumor cell treatment with doxorubicin.

BRCA2 and Smad3 synergize in regulation of gene transcription

Smad3 is an essential component in the intracellular signaling of transforming growth factor-β (TGFβ), which is a potent inhibitor of tumor cell proliferation. BRCA2 is a tumor suppressor involved in early onset of breast, ovarian and prostate cancer. Both Smad3 and BRCA2 possess transcription activation domains. Here, we show that Smad3 and BRCA2 interact functionally and physically. We found that BRCA2 forms a complex with Smad3 in vitro and in vivo, and that both MH1 and MH2 domains of Smad3 contribute to the interaction. TGFβ1 stimulates interaction of endogenous Smad3 and BRCA2 in non-transfected cells. BRCA2 co-activates Smad3-dependent transcriptional activation of luciferase reporter and expression of plasminogen activator inhibitor-1 (PAI-1). Smad3 increases the transcriptional activity of BRCA2 fused to the DNA-binding domain (DBD) of Gal4, and reciprocally, BRCA2 co-activates DBD-Gal4-Smad3. Thus, our results show that BRCA2 and Smad3 form a complex and synergize in regulation of transcription.

Complex of C60 Fullerene with Doxorubicin as a Promising Agent in Antitumor Therapy

The main aim of this work was to evaluate the effect of doxorubicin in complex with C60 fullerene (C60 + Dox) on the growth and metastasis of Lewis lung carcinoma in mice and to perform a primary screening of the potential mechanisms of C60 + Dox complex action. We found that volume of tumor from mice treated with the C60 + Dox complex was 1.4 times less than that in control untreated animals. The number of metastatic foci in lungs of animals treated with C60 + Dox complex was two times less than that in control untreated animals. Western blot analysis of tumor lysates revealed a significant decrease in the level of heat-shock protein 70 in animals treated with C60 + Dox complex. Moreover, the treatment of tumor-bearing mice was accompanied by the increase of cytotoxic activity of immune cells. Thus, the potential mechanisms of antitumor effect of C60 + Dox complex include both its direct action on tumor cells by inducing cell death and increasing of stress sensitivity and an immunomodulating effect. The obtained results provide a scientific basis for further application of C60 + Dox nanocomplexes as treatment agents in cancer chemotherapy.