Melatonin action in tumor skeletal muscle cells: an ultrastructural study.

Melatonin (Mel), or N-acetyl-5-methoxytryptamine, is a circadian hormone that can diffuse through all the biological membranes thanks to its amphiphilic structure, also overcoming the blood-brain barrier and placenta. Although Mel has been reported to exhibit strong antioxidant properties in healthy tissues, studies carried out on tumor cultures gave a different picture of its action, often describing Mel as effective to trigger the cell death of tumor cells by enhancing oxidative stress. Based on this premise, here Mel effect was investigated using a tumor cell line representative of the human alveolar rhabdomyosarcoma (ARMS), the most frequent soft tissue sarcoma affecting childhood. For this purpose, Mel was given either dissolved in ethanol (EtOH) or dimethyl sulfoxide (DMSO) at different concentrations and time exposures. Cell viability assays and ultrastructural observations demonstrated that Mel was able to induce a dose- and time-dependent cell death independently on the dissolution solvent. Microscopy analyses highlighted the presence of various apoptotic and necrotic patterns correlating with the increasing Mel dose and time of exposure. These findings suggest that Mel, triggering apoptosis in ARMS cells, could be considered as a promising drug for future multitargeted therapies.

Hemidesmus indicus induces apoptosis as well as differentiation in a human promyelocytic leukemic cell line.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of the roots of Hemidesmus indicus is widely used in the Indian traditional medicine for the treatment of blood diseases, dyspepsia, loss of taste, dyspnea, cough, poison, menorrhagia, fever, and diarrhea. Poly-herbal preparations containing Hemidesmus are often used by traditional medical practitioners for the treatment of cancer. The aim of this study was to investigate the cytodifferentiative, cytostatic and cytotoxic potential of a decoction of Hemidesmus indicus's roots (0.31-3 mg/mL) on a human promyelocytic leukemia cell line (HL-60). MATERIALS AND METHODS: The decoction of Hemidesmus indicus was characterized by HPLC to quantify its main phytomarkers. Induction of apoptosis, cell-cycle analysis, levels of specific membrane differentiation markers were evaluated by flow cytometry. The analysis of cell differentiation by nitroblue tetrazolium (NBT) reducing activity, adherence to the plastic substrate, α-napthyl acetate esterase activity and morphological analysis was performed through light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: Starting from the concentration of 0.31 mg/ml, Hemidesmus indicus induced cytotoxicity and altered cell-cycle progression, through a block in the G0/G1 phase. The decoction caused differentiation of HL-60 cells as shown by NBT reducing activity, adherence to the plastic substrate, α-naphtyl acetate esterase activity, and increasing expression of CD14 and CD15. The morphological analysis by LM and TEM clearly showed the presence of granulocytes and macrophages after Hemidesmus indicus treatment. CONCLUSIONS: The cytodifferentiating, cytotoxic and cytostatic activities of Hemidesmus indicus offers a scientific basis for its use in traditional medicine. Its potent antileukemic activity provides a pre-clinical evidence for its traditional use in anticancer pharmacology. Further experiments are worthwhile to determine the in vivo anticancer potential of this plant decoction and its components.

AMP-dependent kinase/mammalian target of rapamycin complex 1 signaling in T-cell acute lymphoblastic leukemia: therapeutic implications.

The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic subunit of two multi-protein complexes, referred to as mTORC1 and mTORC2. Signaling downstream of mTORC1 has a critical role in leukemic cell biology by controlling mRNA translation of genes involved in both cell survival and proliferation. mTORC1 activity can be downmodulated by upregulating the liver kinase B1/AMP-activated protein kinase (LKB1/AMPK) pathway. Here, we have explored the therapeutic potential of the anti-diabetic drug, metformin (an LKB1/AMPK activator), against both T-cell acute lymphoblastic leukemia (T-ALL) cell lines and primary samples from T-ALL patients displaying mTORC1 activation. Metformin affected T-ALL cell viability by inducing autophagy and apoptosis. However, it was much less toxic against proliferating CD4(+) T-lymphocytes from healthy donors. Western blot analysis demonstrated dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cells treated with metformin. Remarkably, metformin targeted the side population of T-ALL cell lines as well as a putative leukemia-initiating cell subpopulation (CD34(+)/CD7(-)/CD4(-)) in patient samples. In conclusion, metformin displayed a remarkable anti-leukemic activity, which emphasizes future development of LKB1/AMPK activators as clinical candidates for therapy in T-ALL.

Targeted inhibition of mTORC1 and mTORC2 by active-site mTOR inhibitors has cytotoxic effects in T-cell acute lymphoblastic leukemia.

The mammalian Target Of Rapamycin (mTOR) serine/threonine kinase belongs to two multi-protein complexes, referred to as mTORC1 and mTORC2. mTOR-generated signals have critical roles in leukemic cell biology by controlling mRNA translation of genes that promote proliferation and survival. However, allosteric inhibition of mTORC1 by rapamycin has only modest effects in T-cell acute lymphoblastic leukemia (T-ALL). Recently, ATP-competitive inhibitors specific for the mTOR kinase active site have been developed. In this study, we have explored the therapeutic potential of active-site mTOR inhibitors against both T-ALL cell lines and primary samples from T-ALL patients displaying activation of mTORC1 and mTORC2. The inhibitors affected T-ALL cell viability by inducing cell-cycle arrest in G(0)/G(1) phase, apoptosis and autophagy. Western blot analysis demonstrated a Ser 473 Akt dephosphorylation (indicative of mTORC2 inhibition) and a dephosphorylation of mTORC1 downstream targets. Unlike rapamycin, we found a marked inhibition of mRNA translation in T-ALL cell lines treated with active-site mTOR inhibitors. The inhibitors strongly synergized with both vincristine and the Bcl-2 inhibitor, ABT-263. Remarkably, the drugs targeted a putative leukemia-initiating cell sub-population (CD34(+)/CD7(-)/CD4(-)) in patient samples. In conclusion, the inhibitors displayed remarkable anti-leukemic activity, which emphasizes their future development as clinical candidates for therapy in T-ALL.

Morphological and biochemical patterns in skeletal muscle apoptosis.

Some neuromuscular disorders, such as Duchenne muscular dystrophy, hereditary inclusion body myopathy, malignant hyperthermia, alcoholic myopathy and mitochondrial myopathies are characterized by oxidative stress and loss of muscle fibres due to apoptosis. In this study we have analyzed muscle cell death in vitro utilizing C2C12 myoblasts and myotubes, inducing apoptosis by means of UVB irradiation. C2C12 cells were analysed by scanning and transmission electron microscopy (SEM, TEM) as well as by TUNEL reaction. DNA analysis was performed by gel electrophoresis and flow cytometry. MitoTracker red CMXRos and JC-1 fluorescent probes were also used to study mitochondrial behavior. Finally, caspase activity was investigated by means of Western blot, while caspase-9 and -3 inhibitor effects by means of SEM. SEM showed the typical membrane blebbing while TEM revealed the characteristic chromatin condensation. The TUNEL reaction presented a certain positivity too. Apoptotic and non-apoptotic nuclei in the same myotube were identified both by TUNEL and TEM. Gel electrophoresis never showed oligonucleosomal DNA fragmentation, in agreement with the cell cycle analysis performed by flow cytometry which did not reveal a sharp subdiploid peak. Mitochondrial response to UVB was later investigated and a decrease in mitochondrial functionality appeared. Caspase-9 and -3 cleavage, and, consequently, the activation of the caspase cascade, was also demonstrated by Western blot. Moreover a decrease in apoptotic cell number was noted after caspase-9 and-3 inhibitor treatment. All these results indicated that UVB irradiation induces apoptosis, both in myoblasts and in myotubes, the second being more resistant. DNA fragmentation, at least the nucleosomic type, does not occur. A certain double-strand cleavage appears in TUNEL analysis, as well as characteristic ultrastructural changes in chromatin.

Effects of hyperthermia in human neuroblastoma cells.

Neuroblastoma is a childhood malignancy derived from the developing sympathetic nervous system (SNS) and often diagnosed during early infancy. To investigate its metastatic properties, also in response to anti-cancer treatment, we have studied hyperthermia (HT) effects on the ultrastructure of SK-N-MC human neuroblastoma tumor cells. Cells undergoing HT showed a significant increase in apoptotic and necrotic events, but also a rearrangement of the cellular shape, appearing as cell detachment and rounding. The most striking effects of HT can be so correlated to primary tumor mass decrease and to a certain impairment of cell adhesion properties and consequently metastatic diffusion potential.

Integrin and cytoskeleton behaviour in human neuroblastoma cells during hyperthermia-related apoptosis.

Hyperthermia induces several cellular responses leading to morphological changes, cell detachment and death. Loss of integrins from the cell surface after acute heat-treatment may block several physiological signalling pathways, but whether the assembly network between integrin and cytoskeletal actin is perturbed during hyperthermic treatment is unknown. In this study we tested this hypothesis by evaluating cell morphology, protein cytoskeletal profile and integrin CD11a content in both adherent and floating SK-N-MC human neuroblastoma cells. Morphological and cytometric analyses confirmed that hyperthermia is an effective apoptotic trigger, revealing the typical chromatin margination, cell shape changes and 7-AAD incorporation. After hyperthermia, cytoskeletal proteins showed an increase of high-molecular-weight aggregates and a significant decrease of both actin and CD11a content with respect to control cells. The integrin CD11a and membrane-bound actin alterations found in detached floating neuroblastoma cells recovered after heat-shock may cause the cytoskeletal abnormalities related to the observed surface cell rounding/blebbing and anoikis, early events of hyperthermia-induced programmed cell death.

Hyperthermia triggers apoptosis and affects cell adhesiveness in human neuroblastoma cells.

Hyperthermia is a known apoptotic inducer and has been recently utilized in combination with chemo-and/or radiotherapy in cancer treatment. In this study we have described its effect on SK-N-MC human neuroblastoma tumor cells, a line which grows as a double adherent and floating population. Considering this particular culture behavior, we also investigated the relationship between hyperthermia and cell adhesiveness by evaluating integrin expression, namely CD11a, which is, as known, closely correlated to cell adhesion properties. By a multiple, ultrastructural and flow cytometrical approach, we have demonstrated that hyperthermia, while triggering apoptosis, also determines a CD11a surface expression decrease in apoptotic and living cells. We thus suggest a further role for this treatment, which, affecting adhesion mechanisms, could down-regulate metastatic diffusion.

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds induce apoptotic patterns in cultures of human tumor cell lines.

5-(2-Ethyl-phenyl)-3-(3-methoxy-phenyl)-1H-[1,2,4]triazole (DL-111-IT) and related compounds were extensively studied as anti-gestational agents and some of these molecules were also described as inhibitors of ornithine decarboxylase. Polyamine depletion has been frequently related to the induction of apoptosis and consequently we investigated DL-111-IT and analogs for this effect in myeloid (HL60), neuroblastic (SK-N-MC) and epithelial (BeWo) human tumor cell lines, by means of electron microscopy and DNA electrophoresis. HL60 and SK-N-MC appeared notably sensitive to apoptosis, whereas BeWo responsiveness was variable and frequently associated with necrosis. Our results indicate that the contragestational effect of DL-111-IT and analogs is associated with apoptotic deletion of chorionic tissue and that these molecules, due to their effect on human tumor cell lines, can be considered as antiblastic lead compounds.

Morpho-functional changes in human tendon tissue.

Insertion tissue biopsies of right arm common extensor tendons from 11 patients with chronic lateral epicondylitis were processed for light and electron microscopy. The subjects were aged between 38 and 54 years (only one was 25). The specimens showed a variety of structural changes such as biochemical and spatial alteration of collagen, hyaline degeneration, loss of tenocytes, fibrocartilage metaplasia, calcifying processes, neovascularization and vessel wall modifications. Tissue alterations were evident in limited zones of the tendon fibrocartilage in which the surgical resection was generally visible. The areas where the degenerative processes were localized, were restricted and in spatial contiguity with morphologically normal ones. The observed cases presented histological and electron microscopic findings that characterize lateral epicondylitis as a degenerative phenomenon involving all tendon components.

Actin involvement in apoptotic chromatin changes of hemopoietic cells undergoing hyperthermia.

During apoptosis, cell chromatin undergoes characteristic morphological changes, which have been long described in a variety of experimental models but are mostly not yet understood. The aim of the present study was to investigate the mechanisms underlying this phenomenon and the possible role of cytoskeleton, in particular actin. The chosen apoptotic model were HL60 hemopoietic cells undergoing hyperthermia and the starting point was the observation of thin filament bundles in decondensed chromatin of their early apoptotic nuclei. The characterization of these structures was undertaken by cytochemical, fluorescent and immunogold techniques, directed to actin identification. Taken together, our results suggest, in apoptotic cells, a deep actin rearrangement. Moreover, this cytoskeletal component, never present in normal nucleus, appears in the early apoptotic one, where it can be found in polymerized form, promptly recognizable both by conventional and immunogold electron microscopy. We suggest that, similarly to the role played by nuclear matrix in interphase and mitotic nucleus, actin could be directly involved in chromatin rearrangement occurring in apoptosis.

Giant cell tumor of tendon sheath: a light and electron microscopic study.

A neoformation has been surgically withdrawn from third finger extensor tendon of the right hand of a 52 year male subject. Light (LM) and electron microscope (EM) observations from a number of tissue fragments allowed the identification of tumor nature, which appeared a giant cell tendon sheath. Moreover, some structural patterns have been described and compared to the previously reported cases. In areas of major cell density, parenchyma does not show lobular or gland-like organization; on the other hand, wide zones characterized by an amorphous matrix, progressively replacing collagen and containing elongated cells, are present. Giant multinucleated cells, mostly localized close to collagen bundles, can also be revealed. Unexpectedly, no foam cells appear and no phagocytized cell debris can be identified in giant multinucleated cells. Engulfed crystals are, differently, evidentiated by electron microscopy, both in mono- and multinucleated cell cytoplasm. Their electron density and their localization within cytoplasmic vacuoles suggest the presence of calcium. A correlation between giant cells and osteoclasts is then proposed. Multiple variously oriented centrioles support the possible mitotic genesis of multinucleated giant cells, which never show, on the other hand, fusion features. Siderosomes and residual bodies are also present. An unusual, diffuse thickening of nuclear lamina, only interrupted at nuclear pore level, is described and discussed.

Programmed cell death in 2',3'-dideoxycytidine-resistant human monoblastoid U937 cells.

2',3'-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O2 and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.

Lineage-related sensitivity to apoptosis in human tumor cells undergoing hyperthermia.

In this study the role of hyperthermia as an apoptotic trigger was analyzed in four human tumor cell lines: HL60, U937, DOHH2, and K562. These cell lines were chosen because of their well known and different expression of bcl-2 and bcr-abl genes, the expression of which is known to be an antiapoptotic condition. HL60 and U937 cells were strongly susceptible to heat exposure, while DOHH2 cells were weakly sensitive and K562 cells were resistant, thus suggesting a possible gene involvement in this type of programmed cell death. The mechanisms underlying this apoptosis were investigated by flow cytometry, agarose gel electrophoresis, and light and electron microscopy. A subdiploid peak and DNA laddering, both of which are parameters specifically correlated to programmed cell death, were present in HL60 and U937 and, even if less evident, in DOHH2 cells undergoing hyperthermic treatment, and were absent in K562 cells. In addition, DNA single-strand cleavage was revealed by in situ nick translation, observed by confocal microscopy. Morphological analysis confirmed these results and revealed the typical chromatin changes, followed by the appearance of micronuclei and apoptotic bodies.

Morphological features of apoptosis in hematopoietic cells belonging to the T-lymphoid and myeloid lineages.

Taking into account that apoptosis plays a pivotal role in shaping normal hematopoiesis, morphological features of apoptosis were investigated in both primary cells and continuous cell lines committed towards the T-lymphoid and the myeloid lineages. Apoptosis was induced using: dexamethasone (10(-7) M) for primary rat thymocytes; infection with the T-lymphotropic human herpesvirus 7 (HHV-7) for peripheral blood CD4+ T-cells; staurosporine (1 microM) for MOLT4 CD4+ lymphoblastoid T-cells, HL60 human promyelocytic and U937 human monoblastoid cells; and using senescence of the culture for primary human megakaryocytes. Cell morphology was examined by both transmission electron microscopy and in situ nick translation (NT) revealed by laser scanning confocal microscopy. In spite of the use of different apoptotic agonists, the morphological aspects of apoptosis were similar within the T-lymphoid and the myeloid lineage. While chromatin condensation characterized the early apoptotic events in both lineages, late apoptoses were mainly characterized by further nuclear condensation in lymphoid cells and by production of micronuclei in myeloid cells. Moreover, NT analysis clearly showed that the micronuclei derived from HL60 undergoing apoptosis were composed of both degraded and intact DNA. Thus, T-lymphocytes and myeloid cells showed a lineage-related behavior characterizing the late morphological aspects of apoptosis.

Matrix metalloproteinases expression in HL-60 promyelocytic leukemia cells during apoptosis.

Human promyelocytic leukemia HL-60 cells have been used as a model to study both the expression of matrix-metalloproteinases and the mechanisms of programmed cell death. In the present study we examined the expression of these proteases in HL-60 cells stimulated by different apoptotic triggers. As shown by zymography, HL-60 cells released three major isofroms of the matrix-degrading proteases; when the leukemic cells were grown in serum-free conditions, as well as after hyperthermia and methotrexate treatment, we found a significant loss of the constitutive production of the 92 kDa matrixmetalloprotease, with an unequivocable molecular and ultrastructural evidence of programmed cell death. These results suggest that in HL-60 cells the expression/release of matrix metalloproteases can be down-regulated in the presence of the apoptotic-induced alterations, and that the decreased matrix-degrading capacity of this leukemic cell line during apoptosis may reduce its invasive potential.

Ultrastructural characterization of maturation, platelet release, and senescence of human cultured megakaryocytes.

The aim of this study was to evaluate the ultrastructural features of human megakaryocytes cultured in vitro. For this purpose, pluripotent CD34(+) (cluster of differentiation 34) hematopoietic progenitor cells, obtained from the peripheral blood of healthy adult donors, were differentiated along the megakaryocytic lineage in liquid cultures by the addition of the megakaryocyte-specific growth factor thrombopoietin (TPO, 100 ng/ml). After only 6-8 days, virtually all of the CD34-derived cells expressed the early megakaryocytic CD61 antigen, while, after 15-16 days, most cells also expressed the late megakaryocytic CD42a antigen. Ultrastructural analysis of cells obtained after 7 days of culture showed aspects typical of developing megakaryocytes (MK), such as formation of platelet territories and cytoplasmic fragmentation. At later (15-16 day) culture times, two distinct cell populations were observed: fully developed megakaryocytes releasing platelets into the culture medium and senescent megakaryocytes, characterized by morphological features of apoptosis. Analysis of DNA fragmentation in these cells revealed that apoptosis in megakaryocytes occurred in the absence of the internucleosomic cleavage, which is characteristic of most, but not all, types of apoptosis in cells of hematopoietic origin. On the other hand, flow cytometry of the DNA content of senescent megakaryocytes showed a subdiploid peak that was likely due to a loss of micronuclei during processing.

Apoptosis and necrosis following exposure of U937 cells to increasing concentrations of hydrogen peroxide: the effect of the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide.

A 3-hr exposure of U937 cells to hydrogen peroxide (H2O2) followed by a 6-hr posttreatment incubation in fresh culture medium promotes apoptosis or necrosis, depending on the oxidant concentration. Addition of 3-aminobenzamide (3AB) during the recovery phase prevented necrosis and caused apoptosis. 3AB did not, however, affect the apoptotic response of cells treated with apogenic concentrations of H2O2. Cells exposed for 3 hr to 1.5 mM H2O2, while showing some signs of suffering, maintained a normal nuclear organization and good organelle morphology. At the biochemical level, the oxidant promoted the formation of Mb-sized DNA fragments and rapidly depleted both the adenine nucleotide and non-protein sulphydryl pools, which did not recover during posttreatment incubation in the absence or presence of 3AB. These results allow a novel interpretation of the concentration-dependent switch from apoptosis to necrosis. We propose that H2O2 activates the apoptotic response at the early times of peroxide exposure and that this process can be completed, or inhibited, during the posttreatment incubation phase. Inhibition of apoptosis leads to necrosis and can be prevented by 3AB via a mechanism independent of inhibition of poly(ADP-ribose)polymerase. As a corollary, the necrotic response promoted by high concentrations of H2O2 in U937 cells appears to be the result of specific inhibition of the late steps of apoptosis.

Selective modulation of the cyclin B/CDK1 and cyclin D/CDK4 complexes during in vitro human megakaryocyte development.

Mammalian megakaryocyte development is characterized by a progressive accumulation of cells exhibiting a polylobated nucleus with a polyploid DNA content. In this study human megakaryocytes were obtained from CD34+ haemopoietic progenitors by in vitro liquid culture in the presence of 100 ng/ml of recombinant thrombopoietin (TPO). Ultrastructural examination of polyploid megakaryocytes showed the presence of a large number of centrioles, the breakdown of the nuclear envelope, and the progressive chromatin condensation, all aspects characteristic of mitosis. At both indirect immunofluorescence and Western blot analyses, cyclin B and its related cyclin-dependent kinase (CDK)1, which forms the mitosis promoting factor (MPF), showed an increased expression in maturating megakaryoblasts and megakaryocytes (day 8 of culture) with respect to freshly isolated CD34+ progenitors. This expression tended to decline in fully developed megakaryocytes (day 15 of culture). The amount of cyclin D and of the related CDK4, governing the G1 phase of the cell cycle, increased during megakaryocyte development, maintaining high levels of expression also in mature megakaryocytes. These results indicate that megakaryocyte polyploidization depends on a true, although incomplete, mitotic process, and that cyclin D/CDK4 probably plays a crucial role throughout megakaryocytopoiesis.

Apoptotic pathways depend on the target enzymatic activity and not on the triggering agent.

Molt-4 human leukemia cells were triggered to apoptosis by various agents with different mechanisms of action. Staurosporine, a protein kinase C (PKC) inhibitor; camptothecin, a topoisomerase I blocking drug; and tiazofurin, an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH), were used. Ultrastructural analysis showed morphologic changes characteristic of apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotic agents with different mechanisms of action induced the formation of large genomic DNA fragments and very similar ultrastructural changes. Therefore, both phenomena and the closely related apoptosis progression depend on target cell machinery and not on the triggering agent.