Emil Rudolf

Zinc pyrithione induces cellular stress signaling and apoptosis in Hep-2 cervical tumor cells: the role of mitochondria and lysosomes

Increased intracellular free zinc concentrations are associated with activation of several stress signaling pathways, specific organelle injury and final cell death. In the present work we examined the involvement of mitochondria and lysosomes and their crosstalk in free zinc-induced cell demise. We report that treatment of cervical tumor Hep-2 cells with zinc pyrithione leads to an early appearance of cytoplasmic zinc-specific foci with corresponding accumulation of zinc first in mitochondria and later in lysosomes. Concomitant with these changes, upregulation of expression of metallothionein II A gene as well as the increased abundance of its protein occurs. Moreover, zinc activates p53 and its dependent genes including Puma and Bax and they contribute to an observed loss of mitochondrial membrane potential and activation of apoptosis. Conversely, lysosomal membrane permeabilization and its promoted cleavage of Bid occurs in a delayed manner in treated cells and their effect on decrease of mitochondrial membrane potential is limited. The use of specific inhibitors as well as siRNA technology suggest a crucial role of MT-IIA in trafficking of free zinc into mitochondria or lysosomes and regulation of apoptotic or necrotic cell demise.

Far-Red-Absorbing Cationic Phthalocyanine Photosensitizers: Synthesis and Evaluation of the Photodynamic Anticancer Activity and the Mode of Cell Death Induction

Novel zinc, magnesium, and metal-free octasubstituted phthalocyanine photosensitizers bearing [(triethylammonio)ethyl]sulfanyl substituents in the peripheral or nonperipheral positions were synthesized and investigated for their photophysical properties (ΦΔ value up to 0.91, λmax up to 750 nm) and photodynamic anticancer activity. The photodynamic treatment of 3T3, HeLa, SK-MEL-28, and HCT 116 cancer cells revealed that the magnesium complexes were not active (IC50 > 100 μM), whereas the IC50 values of the zinc complexes typically reached values in the submicromolar range with low toxicity in the dark (TC50 ≈ 1500 μM). The subcellular changes upon photodynamic treatment of the HeLa cells indicated that the studied photosensitizers induced damage primarily to the lysosomes, which was followed by a relocalization and damage to other organelles. The time-lapse morphological changes along with the flow cytometry and caspase activity measurements indicated a predominant involvement of necrosis-like cell death.

Synthesis, Properties and In Vitro Photodynamic Activity of Water‐soluble Azaphthalocyanines and Azanaphthalocyanines

In this work zinc azaphthalocyanines (AzaPcs) from the group of tetrapyrazinoporphyrazines and zinc azanaphthalocyanines from the group of tetra[6,7]quinoxalinoporphyrazines (TQP) with eight diethylaminoethylsulfanyl substituents were synthesized. Tertiary amines were later quaternized with ethyl iodide to obtain water‐soluble photosensitizers (PSs). Quaternized compounds showed high singlet oxygen quantum yields as determined in DMF by monitoring decomposition of 1,3‐diphenylisobenzofuran. In water medium, quaternized AzaPc derivatives appeared in monomeric form in a wide range of concentrations while quaternized TQP derivatives showed aggregation at higher concentrations (over 1 μm). Photodynamic activity was tested on Hep2 cells using light of λ > 640 nm. Both quaternized dyes showed high photodynamic activity (IC50 = 104 and 220 nm for AzaPc and TQP, respectively). Dark toxicity was not detected even at the highest concentration used in in vitro tests (200 μm) which indicates a promising therapeutic index of these new substances. Tested compounds localized inside the cells mainly within the lysosomes thus suggesting an endocytic mechanism of cellular uptake. No localization within mitochondria was detected. A great advantage of TQP derivatives over other PSs is their very strong absorption at 747 nm that allows activation at wavelengths penetrating deeper into human tissues.

Polyphenolic Coumpounds in Chemoprevention of Colon Cancer - Targets and Signaling Pathways

Colorectal cancer is one of the leading causes of premature death in people worldwide. Due to the fact that malignant conversion of normal colonic cells requires several steps and often proceeds over considerable time periods, primary prevention of this process should include several approaches, with optimization of nutrition and diet being among most important. During past decades, several groups of chemicals (both naturally occurring as well as synthetic) have been studied in terms of their potential chemopreventive role in colorectal cancer development. Naturally occurring plant polyphenols have recently come into scientific focus because of their presence in various popular natural products (wine grapes, teas, berries, peanuts) and, more importantly, due to their reported antiproliferative and cytostatic abilities in various in vitro and in vivo models. This review seeks to summarize the currently known targets and mechanisms whereby polyphenolic compounds interfere with colonic cancer cells while evaluating their chemopreventive potential in vivo.

Interaction of B cells with intracellular pathogen Francisella tularensis

Immunity to Francisella tularensis is largely mediated by T lymphocytes but an important role of B lymphocytes in early stage of infection was previously uncovered. We wanted to find out if F. tularensis is able to infect B cells and/or influence them by direct contact. To investigate this possibility we infected B cell lines from mouse (A20) or humans (Ramos RA-1), or primary mouse spleen cells, with F. tularensis LVS and F. tularensis FSC200 in vitro. In all cases, we detected bacteria on the cell surface and inside the B cells using transmission electron microscopy. More than 20% cells were infected by microbes after 24h. The number of bacteria, determined by CFU, increased about 1 log during 24h. Infection with live bacteria led to apoptosis of Ramos cells and mouse CD19(+) spleen cells. Approximately 30% of cells were apoptotic after 24h and 70% after 48 h, independently of the F. tularensis strain, while only 10% of non-infected cell were apoptotic at either time point. Apoptosis was confirmed by Western blot using anti-PARP antibodies. Thus, this study demonstrates unique phenomenon - namely, the ability of the intracellular pathogen F. tularensis to invade and induce apoptosis in B cells.

Selenite-induced apoptosis and autophagy in colon cancer cells.

Sodium selenite (Se) is known to induce diverse stress responses in malignant cells which may lead to various types of cell death including apoptosis and/or autophagy. In colon cancer cells, Se activates several signaling pathways whose interactions and ultimate endpoints may vary in individual study models. In our previous work we showed differences in Se-dependent growth inhibition, cell cycle alterations and apoptosis in colon cancer cells with functional (HCT-116) and deleted (HCT-116-p53KO) p53. Moreover, detailed morphological and biochemical analyses revealed the presence of autophagy in Se-treated cells. Thus the aim of this study was to investigate in detail mechanisms, relationship and crosstalk between apoptosis and autophagy in Se-treated HCT-116 cancer cells differing in p53 status since p53 has been shown to play a well-known role in apoptosis but dichotomous role in autophagy. We report that the absence of p53 in malignant colonocytes changes patterns of response to Se-induced stress which include differential activation of MAP kinases (p38 - HCT-116 and JNK - HCT-116 p53KO) including their respective roles in the process of apoptosis and autophagy as well as the involvement of mTOR or PI3K signaling. Our results seem to suggest that deletion of p53 inevitably leads to a higher level of instability and delays in an individual cell decision in the face of stress whether to activate apoptosis or autophagy which may consequently occur simultaneously with mutual dichotomous relationship.

Proliferation and Differentiation of Adult Endogenous Neural Stem Cells in Response to Neurodegenerative Process within the Striatum

The ongoing process of neurogenesis in the adult mammalian forebrain suggests the possible capacity for limited self-repair after brain injury. Previously, we have demonstrated that in an animal model of Huntington’s disease the neurodegenerative process initiates immediate intensive cell proliferation and differentiation resulting in characteristic enlargement of the subependymal zone (SEZ) of lateral brain ventricles. Now, our interest is focused on the architecture of the neurogenic niche of the SEZ in the identical model, particularly on characteristic features of astrocyte-like cells which are considered to be not only niche cells but also neural stem cells. Our findings prove higher activation of the lateral part of the SEZ (L-SEZ) adjacent to the degenerated striatum compared with the rostral part of the SEZ (R-SEZ). In the activated L-SEZ, niche cells which ensheathe clusters of neural progenitors are of immature astrocytic phenotype because of nestin and vimentin expression (except the expression of glial fibrillary acidic protein). However, the coexpression of all three filaments is not always found. Intermediate filaments also enable us to distinguish the basic shape of astrocytic cells within the SEZ, majority of which resemble protoplasmic rather than fibrillary astrocytes. Furthermore, our results show a wide plasticity of these astrocyte-like cells in immediate response to an extensive pathological process in the brain. These observations are consistent with the fact that adult stem cells undergo different processes in an already mature environment, and therefore can exhibit some specific characteristics unlike the embryonic or fetal neural stem cells.

Activation of B cell apoptotic pathways in the course of Francisella tularensis infection.

Francisella tularensis is a facultative intracellular, gram-negative bacterium that induces apoptosis in macrophages and B cells. Here we show apoptotic pathways that are activated in the Ramos human B cell line in the course of F. tularensis infection. Live bacteria F. tularensis FSC200 activate caspases 8, 9 and 3, as well as Bid; release cytochrome c and apoptosis-inducing factor from mitochondria; and induce depolarization of mitochondrial membrane potential in the Ramos cell line, thus leading these cells to apoptosis. Unlike live bacteria, killed F. tularensis FSC200 bacteria activated only caspase 3, and did not cause apoptosis of Ramos cells as measured by annexin V. Killed bacteria also caused accumulation of anti-apoptotic protein Bclx(L) in mitochondrial membranes. Thus, live F. tularensis activates both caspase pathways (receptor-mediated and intrinsic) as well as caspase-independent mitochondrial death.

Sulforaphane induces cytotoxicity and lysosome- and mitochondria-dependent cell death in colon cancer cells with deleted p53

Mechanisms and pathways responsible for cytotoxicity of sulforaphane (SF) in colon cancer cells with deleted p53 were investigated during 48 h of exposure. SF showed dose-dependent cytotoxicity and proapoptotic activity in the present model. In addition, in HCT-116 p53KO cells SF induced DNA damage with the subsequent cellular response and signaling not including p53 and caspase-2 pathways. Conversely, in SF-treated cells JNK was activated which led to an early lysosomal membrane permeabilization, release of cathepsin B and D and activation of Bid by specific cleavage. Concomitantly, the expression of Bax increased in the presence of JNK-mediated Bcl-2 inhibition which was followed by mitochondrial release of cytochrome c and activation of apoptosis. These results suggest that SF may be useful as a chemopreventive agent in colon cancer with inactivated or lost p53.

Camptothecin induces p53-dependent and -independent apoptogenic signaling in melanoma cells

Various DNA-targeting agents may initiate p53-dependent as well as p53-independent response and subsequent apoptosis via alternative cellular systems which include for instance p73, caspase-2 or Bcl-2 family proteins. The scope of involvement of individual molecules in this process and the mechanisms governing their potential interplay are still not entirely understood, in particular in highly aggressive cancers such as in malignant melanoma. In this work we investigated the role and involvement of both p53-dependent and -independent mechanisms in selected melanoma cell lines with differing status of p53 using a model DNA topoisomerase I inhibitor camptothecin (CPT). Here we report that CPT induced in Bowes melanoma cells apoptosis which is essentially p53 and mitochondria-dependent but with some involvement of caspase-2 and p73. Conversely, in mutant p53 melanoma cells overall levels of CPT-induced apoptosis are significantly lower, with p73 and caspase-2 signaling playing important roles. In addition, in these cells the expression of micro RNAs family 34 (miR-34) were low compared to wild-type p53 cells. The ectopic expression of wild type p53 than restored apoptotic response of cells to CPT despite the fact that the expression of miR-34 and miR-155 were not influenced. These results suggest that CPT induces multivariate cellular stress responses including activation of DNA-damage response-p53 pathway as well as p53-independent signaling and their mutual crosstalk play the decisive role in the efficient triggering of apoptosis in melanoma cells.