The effect of 3-aminobenzamide, inhibitor of poly(ADP-ribose) polymerase, on human osteosarcoma cells

This study demonstrates that in human osteosarcoma cells treatment with 3-aminobenzamide (3-AB), a potent inhibitor of poly(ADP-ribose) polymerase (PARP), induces morphological and biochemical features of differentiation, the duration of which depends on whether or not the normal RB gene is expressed. In Saos-2 cells expressing a non-functional Rb protein, 3-AB treatment induced the formation of transient, short dendritic-like protrusions. In RB-transfected-Saos-2 cells (a clone previously generated in our laboratory that shows stable expression of wild-type Rb protein), 3-AB induced marked and prolonged changes with the formation of long dendritic-like protrusions and the appearance of ste…

Apoptosis induced in HepG2 cells by the synthetic cannabinoid WIN: involvement of the transcription factor PPARgamma.

It has recently been shown that cannabinoids induce growth inhibition and apoptosis in different tumour cell lines. In the current study, the effects of WIN 55,212-2 (WIN), a synthetic and potent cannabinoid receptor agonist, are investigated in hepatoma HepG2 cells and a possible signal transduction pathway is proposed. In these cells, WIN induces a clear apoptotic effect which was accompanied by up-regulation of the death-signalling factors Bax, Bcl-X(S), t-Bid and down-regulation of the survival factors survivin, phospho-AKT, Hsp72 and Bcl-2. Moreover, WIN-induced apoptosis is associated with JNK/p38 MAPK pathway activation and mitochondrial depolarisation demonstrated by a cytofluorimet…

pRb suppresses camptothecin-induced apoptosis in human osteosarcoma Saos-2 cells by inhibiting c-Jun N-terminal kinase

AbstractThis paper studies the cytotoxic effect induced by the topoisomerase I inhibitor camptothecin in human osteosarcoma Saos-2 cells, which lack p53 and contain a non-functional form of the product of the retinoblastoma gene, pRb. Cytotoxicity induced by camptothecin was dose- and time-dependent; the treatment with 100 nM camptothecin reduced cell viability by 50% at 32 h and by 75% at 72 h of exposure. The cytotoxic effect was caused by apoptosis, as ascertained by morphological evidence, acridine orange-ethidium bromide staining and flow cytometric analysis. Apoptosis was accompanied by both the activation of caspase-3 and the fragmentation of poly(ADP-ribose) polymerase. Treatment wi…

The Synergistic Effect of SAHA and Parthenolide in MDA-MB231 Breast Cancer Cells

The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity…

Role of PPARγ in apoptosis induced by cannabinoids in hepatoma HepG2 cells.

Differentiative pathway activated by 3-aminobenzamide, an inhibitor of PARP, in human osteosarcoma MG-63 cells

AbstractThis study describes the molecular mechanism by which treatment with 3-AB, a potent inhibitor of PARP, allows human osteosarcoma MG-63 cells to restrict growth and enter differentiation. Our findings show that in MG-63 cells, aberrant gene expression keeps Rb protein constitutively inactivated through hyperphosphorylation and this promotes uncontrolled proliferation of the cells. After 3-AB-treatment, the poly(ADP-ribosyl)ation of nuclear proteins markedly decreases and this results in an increase in both the hypophosphorylated active form of Rb and pRb/E2F complexes. These effects are accompanied by G1 arrest, downregulation of gene products required for proliferation (cyclin D1, β…

The synthetic cannabinoid WIN55,212-2 synergizes with the death receptor ligand TRAIL to induce apoptotic effect in HepG2 hepatoma cells.

The effects of glucocorticoids on thymidine kinase and nucleoside phosphotransferase during development of chicken embryo retina.

AbstractThymidine kinase in chick embryo retina reaches its highest values on the 8–10th day of development, then declines reaching the lowest value at hatching. The rate of DNA synthesis essentially follows this activity while, in contrast, nucleoside phosphotransferase increases progressively during development. Glucocorticoids at 5 × 10−6M lower the level of thymidine kinase in isolated retinas of chick embryo. The most effective steroid was hydrocortisone. The effect was observed in retinas from 8–18-day-old chick embryo and, except on the 18th day, was always of the same magnitude. We suggest that a glucocorticoid can be the natural factor responsible for the marked fall in thymidine k…

RB1 in cancer: Different mechanisms of RB1 inactivation and alterations of pRb pathway in tumorigenesis

Loss of RB1 gene is considered either a causal or an accelerating event in retinoblastoma. A variety of mechanisms inactivates RB1 gene, including intragenic mutations, loss of expression by methylation and chromosomal deletions, with effects which are species-and cell type-specific. RB1 deletion can even lead to aneuploidy thus greatly increasing cancer risk. The RB1gene is part of a larger gene family that includes RBL1 and RBL2, each of the three encoding structurally related proteins indicated as pRb, p107, and p130, respectively. The great interest in these genes and proteins springs from their ability to slow down neoplastic growth. pRb can associate with various proteins by which it …

Loss of MCL1 function sensitizes the MDA-MB-231 breast cancer cells to rh-TRAIL by increasing DR4 levels.

Triple-negative breast cancer (TNBC) is a form of BC characterized by high aggressiveness and therapy resistance probably determined by cancer stem cells. MCL1 is an antiapoptotic Bcl-2 family member that could limit the efficacy of anticancer agents as recombinant human tumor necrosis factor related apoptosis-inducing ligand (rh-TRAIL). Here, we investigated MCL1 expression in TNBC tissues and cells. We found MCL1 differentially expressed (upregulated or downregulated) in TNBC tissues. Furthermore, in comparison to the human mammary epithelial cells, we found that MDA-MB-231 cells show similar messenger RNA levels but higher MCL1 protein levels, whereas it resulted downregulated in MDA-MB-…

Nucleoside phosphotransferase of chick embryo

This paper describes a purification procedure and some properties of a nonspecific nucleoside phosphotransferase of chick embryo, an activity which catalyzes the transfer of chick embryo, an activity which catalyzes the transfer of the phosphate ester from a deoxyribonucleotide or a pyrimidine ribonucleotide to a deoxyribonucleoside acceptor. The enzyme is very unstable to heat, dilution and dialysis and it is almost entirely inactivated by DEAE-cellulose chromatography or gel filtration. A marked enhancement in its stability is caused by numerous nucleotides. In these experiments at least 920-fold purification was obtained by using dTTP (50 microM) as nucleotide protector. The enzyme, puri…

Apoptotic effects of different drugs on cultured retinoblastoma Y79 cells

This paper deals with the apoptotic effect exerted in human retinoblastoma Y79 cells by a number of compounds. A remarkable effect was observed after treatment with DNA-damaging agents, such as camptothecin, etoposide, cisplatin and carboplatin; camptothecin was found to be the most efficacious. Treatment with these compounds induced the appearance of morphological features of apoptosis in the cells together with the distinct fragmentation of DNA, as shown by agarose gel electrophoresis. These effects were also accompanied by a remarkable increase in the level of p53. Many other compounds, which are not DNA-damaging agents, induced the morphological features of apoptosis but none of them we…

In human osteosarcoma 3AB-OS cancer stem cells let-7d seems to have the dual function of oncogene/tumor-suppressor miRNA

Introduction: Osteosarcoma (OS), an aggressive tumor a!ecting adolescents, shows therapy resistance and recurrence which can depend on cancer stem cells (CSCs), the source for tissue renewal and hold malignant potential; thus, OS treatment requires their eradication. Here, using 3AB-OS CSCs previously obtained from the OS-MG63 cells, we focused on the role of let-7d in managing stemness properties of 3AB-OS CSCs. Methods: 3AB-OS CSCs were stably transfected with pCDomH-plasmid, containing mir-let7-d, or empty vector as a control. Cell proliferation and viability were evaluated by EdU and Trypan blue assays, respectively. Sarcosphere and colony assays and wound healing and transwell invasion…

Composizioni farmaceutiche per il trattamento di tumori epatici

Let-7d miRNA Shows Both Antioncogenic and Oncogenic Functions in Osteosarcoma-Derived 3AB-OS Cancer Stem Cells

Osteosarcoma (OS), an aggressive highly invasive and metastatic bone-malignancy, shows therapy resistance and recurrence, two features that likely depend on cancer stem cells (CSCs), which hold both self-renewing and malignant potential. So, effective anticancer therapies against OS should specifically target and destroy CSCs. We previously found that the let-7d microRNA was downregulated in the 3AB-OS-CSCs, derived from the human OS-MG63 cells. Here, we aimed to assess whether let-7d modulation affected tumorigenic and stemness properties of these OS-CSCs. We found that let-7d-overexpression reduced cell proliferation by decreasing CCND2 and E2F2 cell-cycle-activators and increasing p21 an…

Role of insulin-like growth factors in autocrine growth of human retinoblastoma Y79 cells.

In this study, we have demonstrated that human retinoblastoma Y79 cells produce insulin-like growth factors (IGFs) type I and type II and release them into the medium. We have also ascertained, by means of competitive studies and cross-linking procedure, that Y79 cells contain the type-I IGF receptor (IGF-IR). Furthermore, surface-bound IGF-I is internalised by the receptor, then degraded to amino acids. Insulin, IGF-I and IGF-II caused down-regulation of IGF-IR; the effect is concentration and time dependant. Scatchard analysis demonstrated that incubation with insulin markedly decreased the binding capacity measured for IGF-I while the apparent Kd value calculated for IGF-I binding was no…

Insulin and IGFs induce apoptosis in chick embryo retinas deprived of L-glutamine

In chick embryo retinas, cultured in serum-free medium lacking L-glutamine, IGF-I, IGF-II and insulin induced apoptotic DNA fragmentation and cell death, IGF-I being the most efficacious compound. The apoptotic effect, which was particularly evident in retinas removed from 7-day-old chick embryos, declined with the age of the embryos and disappeared after day 11. Apoptosis appeared after a time lag of 8 h and then increased with time up to 16 h. Cycloheximide, an inhibitor of protein synthesis, was capable of entirely abolishing apoptotic cell death. The effect induced by IGFs or insulin was suppressed by the addition of glutamine. Cytokine-mediated apoptosis was also observed after withdra…

Parthenolide induces EGF receptor phosphorylation and superoxide anion production in MDA-MB231 breast cancer cells.

The proteasome inhibitor bortezomib in programmed cell death and cancer therapy

The 26S proteasome is a ubiquitous enzyme complex which is responsible for degrading proteins that regulate essential cellular functions, such as cell-cycle control, cellular adhesion, proliferation, apoptosis and angiogenesis. The inhibition of proteasome dysregulates these signalling pathways, ultimately leading to cell cycle arrest and apoptosis in many cell types. Importantly, preclinical research has shown that cancer cells seem to be more sensitive to the proapoptotic effects of proteasome inhibition than normal cells. First-generation proteasome inhibitors lacked usefulness because of broad specificity and irreversible binding to the proteasome. However, the peptide boronic acid prot…

Inhibition of DNA synthesis in chick embryo retinas, in vitro, by a factor from fetal bovine serum

Fetal bovine serum inhibited deoxyribonucleic acid (DNA) synthesis in chick embryo retina explants. The inhibitory activity was precipitated from fetal bovine serum by 45% saturated ammonium sulfate and isolated by means of Sephadex G-100 and Bio-Gel P-60 columns as a peak with an apparent molecular weight of 7000 Da. DNA-inhibiting activity was heat- and acid-stable and was destroyed by dithiothreitol and alkaline treatment. The purified factor inhibited similarly both DNA synthesis and thymidine kinase activity; 50% inhibitory effect was found with 160 ng, 17 h after the addition into the incubation medium.

L’incertezza nella valutazione dell’efficacia degli interventi di adeguamento

The mechanism by which histone deacetylase inhibitors sensitize hepatoma and colon cancer cells to TRAIL-induced apoptosis

Proteasome inhibitor Velcade induces apoptosis in hepatoma HepG2 cells stimulating both the extrinsic and the intrinsic apoptotic pathways.

Morphological and biochemical effects of glucocorticoids in chick embryo hepatocytes during development

Abstract The administration in ovo of hydrocortisone-21-phosphate caused, in chick embryo liver, a reduction of the number of hepatocytes which can be isolated from 1 mg dry weight of liver and a marked increase of their size. Moreover, the treatment diminished the incorporation of thymidine into acid-insoluble fraction in these cells whilst it augmented the content of protein, RNA, DNA and the level of thymidine kinase/cell. These effects were highest at 8–10 days, then declined with the age, disappearing after 18th day of incubation. Similar effects were obtained by injecting other glucocorticoids or ACTH. Combined treatment with metopirone abolished the effects found with ACTH, but did n…

Apoptosis induced in hepatoblastoma HepG2 cells by the proteasome inhibitor MG132 is associated with hydrogen peroxide production, expression of Bcl-Xs and activation of caspase-3

This report is focused on the apoptotic effect induced by MG132, an inhibitor of 26S proteasome, in human hepatoma HepG2 cells. The results were compared with those obtained with non-transformed human Chang liver cells. MG132 reduced the viability of HepG2 cells in a time- and dose-dependent manner. The effect was in tight connection with the induction of apoptosis, as indicated by fluorescence microscopy and cytometric analysis, and was accompanied by a remarkable increase in the production of H2O2 and a reduction in mitochondrial transmembrane potential (Deltapsim). In addition cell death was prevented by antioxidants such as GSH, N-acetylcysteine or catalase. Western blot analysis showed…

Parthenolide prevents resistance of MDA-MB231 cells to doxorubicin and mitoxantrone: the role of Nrf2.

Triple-negative breast cancer is a group of aggressive cancers with poor prognosis owing to chemoresistance, recurrence and metastasis. New strategies are required that could reduce chemoresistance and increases the effectiveness of chemotherapy. The results presented in this paper, showing that parthenolide (PN) prevents drug resistance in MDA-MB231 cells, represent a contribution to one of these possible strategies. MDA-MB231 cells, the most studied line of TNBC cells, were submitted to selection treatment with mitoxantrone (Mitox) and doxorubicin (DOX). The presence of resistant cells was confirmed through the measurement of the resistance index. Cells submitted to this treatment exhibit…

JNK and AP-1 mediate apoptosis induced by bortezomib in HepG2 cells via FasL/caspase-8 and mitochondria-dependent pathways

The proteasome inhibitor bortezomib is an efficacious apoptotic agent in many tumor cells. This paper shows that bortezomib induced apoptosis in human hepatoma HepG2 cells associated with many modifications in the expression of survival or death factors. Although bortezomib increased the level of the protective factors HSP70 and HSP27, the effects of the drug that favour cell death were predominant. These events include accumulation of c-Jun, phospho-c-Jun and p53; increase in FasL level with activation of caspase-8; changes related to members of Bcl-2 family with increase in the level of pro-apoptotic members and decrease in that of anti-apoptotic ones; dissipation of mitochondrial potenti…

Differentiation of human osteosarcoma 3AB-OS stem-like cells in derivatives of the three primary germ layers as an useful in vitro model to develop several purposes

A number of solid tumors contain a distinct subpopulation of cells, termed cancer stem cells (CSCs) which represent the source for tissue renewal and hold malignant potential and which would be responsible for therapy resistance. Today, the winning goal in cancer research would be to find drugs to kill both cancer cells and cancer stem cells, while sparing normal cells. Osteosarcoma is an aggressive pediatric tumor of growing bones that, despite surgery and chemotherapy, is prone to relapse. We have recently selected from human osteosarcoma MG63 cells a cancer stem-like cell line (3AB-OS), which has unlimited proliferative potential, high levels of stemness-related markers, and in vivo tumo…

Induction of apoptosis by arachidonic acid in human retinoblastoma Y79 cells: involvement of oxidative stress

Arachidonic acid administration caused apoptosis in Y79 cells, as shown by typical morphological changes, phosphatidylserine externalization, chromatin condensation, processing and activation of caspase-3 and cleavage of the endogenous caspase substrate poly-(ADP-ribose)-polymerase. Arachidonic acid also caused lamin B cleavage, suggesting caspase-6 activation. Arachidonic acid treatment was accompanied by increased formation of the lipid peroxidation end products malondialdehyde and 4-hydroxy-2-nonenal, lowering in reduced glutathione content and in mitochondrial membrane potential. Inhibiting glutathione synthesis sensitized Y79 cells to apoptosis-inducing stimuli, whilst replenishing red…

Parthenolide sensitizes human hepatocarcinoma cells to TRAIL-induced apoptosis

Genetic and Molecular Characterization of The Human Osteosarcoma 3AB-OS Cancer Stem Cell Line: A Possible Model For Studying Osteosarcoma Origin and Stemness

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second-line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB-OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB-OS cells have hypertriploid karyotype wit…

MicroRNA-29b-1 impairs in vitro cell proliferation, self‑renewal and chemoresistance of human osteosarcoma 3AB-OS cancer stem cells

Osteosarcoma (OS) is the most common type of bone cancer, with a peak incidence in the early childhood. Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. MicroRNAs (miRNAs) have been implicated in the maintenance of the CSC phenotype, thus, identification of CSC-related miRNAs would provide information for a better understanding of CSCs. Downregulation of miRNA-29 family members (miR-29a/b/c; miR‑29s) was observed in human OS, however, little is known about the functions of miR-29s in human OS CSCs. Previously, during the characterization of 3AB-OS cells, a CSC line selected from human OS MG63 cells, we…

Chick embryo retina development in vitro: the effect of insulin.

In this paper we study the development of chick embryo retina cultured in vitro and the effects exerted by insulin. Retinas were removed from 7-day embryos and cultured in serum- and hormone-free medium for 7 additional days. Under these conditions retinal cells survived and underwent cholinergic differentiation, as previously ascertained by Hausman et al. (Dev. Brain Res., 1991, 59: 31-37). However, a great retardation of development was noted compared to uncultured control, 14-day retina. In fact both wet weight and DNA and protein content increased much slower than in ovo and the tubulin content decreased below even the starting value. In addition, although after 7 days in culture retina…

Identification and expansion of human osteosarcoma-cancer-stem cells by long-term 3-aminobenzamide treatment

A novel cancer stem-like cell line (3AB-OS), expressing a number of pluripotent stem cell markers, was irreversibly selected from human osteosarcoma MG-63 cells by long-term treatment (100 days) with 3-aminobenzamide (3AB). 3AB-OS cells are a heterogeneous and stable cell population composed by three types of fibroblastoid cells, spindle-shaped, polygonal-shaped, and rounded-shaped. With respect to MG-63 cells, 3AB-OS cells are extremely smaller, possess a much greater capacity to form spheres, a stronger self-renewal ability and much higher levels of cell cycle markers which account for G1-S/G2-M phases progression. Differently from MG-63 cells, 3AB-OS cells can be reseeded unlimitedly wit…

Role of the acetylation of p53 and histones in SAHA-induced apoptosis in human hepatoma HepG2 cells.

The cytotoxic effect exerted by parthenolide and DMAPT on breast cancer stem-like cells

Triple-negative breast cancers (TNBCs) are aggressive forms of breast carcinoma associated with a high rate of recidivism. It is known that a small proportion of tumour cells, termed cancer stem cells (CSCs), is responsible for tumour formation, progression and recurrence. The sesquiterpene lactone parthenolide (PN) was identified as the first small molecule capable of killing CSCs.1 Previously we have shown2 that PN and its soluble analogue DMAPT induce a strong cytotoxic effect in MDA-MB231 cells, the most studied line of TNBCs. In the present research we investigated about the effects exerted by both PN and DMAPT on breast cancer stem-like cells derived from three lines of TNBCs (MDA-MB2…

Insulin synthesis in chick embryo retinas during development.

Retinas of chick embryos contain insulin (1) and further, are capable of synthesizing it, as demonstrated by incubating retinas at different ages (7th–18th day) with [3H]leucine. The synthesized radioactive insulin was isolated and assayed by means of a HPLC procedure. The synthesis of insulin was found to be highest in the youngest retinas studied (day 7), afterwards it declined with age except for an increment found at 14–15 day. Explants of chick embryo retinas, cultured in vitro, rapidly degraded insulin. Nevertheless, the content of immunoreactive insulin in retinal explants diminished slowly with the age of culture, so that, after 8 days of incubation, it was about 60% of the content …

Histone deacetylase inhibitors: Epigenetic drugs acting by pleiotropic apoptotic mechanism in tumor cells and highly potent in combination with other antitumor agents.

Histone deacetylase inhibitors induce in human hepatoma HepG2 cells acetylation of p53 and histones in correlation with apoptotic effects

This report shows that histone deacetylase inhibitors (HDACIs) induced apoptosis in human hepatoma HepG2 cells in a dose- and time-dependent manner. Trichostatin A (TSA), ITF2357 and suberoylanilide hydroxamic acid (SAHA), which were very effective agents, caused apoptotic effects after a lag phase of 12-16 h. In order to elucidate the mechanism of HDACIs action in HepG2 cells we have studied the effects of TSA, ITF2357 and SAHA on acetylation of p53 and histones H2A, H2B, H3 and H4. It was observed that HDACIs rapidly induced acetylation of these proteins, being the effects clearly visible already at 30 min of treatment at the same doses which caused apoptosis. Analysis of the immunocomple…

Parthenolide induces caspase-independent cell death mediated by AIF in osteosarcoma and melanoma cells.

Parthenolide, the major bioactive sesquiterpene lactone present in Feverfew (Tanacetum parthenium), has recently attracted considerable attention because of its complex pharmacological action involving anti-microbial, anti-inflammatory and anti-cancer effects. However, the mechanism of its cytotoxic effect on tumor cells still remains scarcely defined today. The aim of this study was to analyse the mechanism of parthenolide action on two lines of cancer cells, the human osteosarcoma MG63 and the melanoma SK-MEL-28 cells, on which parthenolide exerted its action inducing similar effects. Staining with Hoechst 33342 showed that parthenolide induced in the first phase of treatment (0-5 h) in m…

Verifica sperimentale sul comportamento delle pavimentazioni stradali di tipo flessibile rinforzate con rete metallica all'azione tangenziale dei carichi

Synthesis of insulin and its effects in Y79 human retinoblastoma cells

This paper demonstrates that Y79 human retinoblastoma cells contain immunoreactive insulin (IRI) and release it in the medium. Cells cultured either in suspension or in monolayer showed a similar content of IRI. Moreover, in both conditions, IRI concentration was higher in cells cultured in serum-supplemented medium rather than in serum-free medium. Retinoblastoma cells are capable of synthesizing insulin. This was demonstrated by incubating Y79 cells with [3H]leucine. The synthesized radioactive insulin was separated and assayed by means of a HPLC procedure described in this paper. Both cell growth and [3H]thymidine and [3H]uridine incorporation into acid-insoluble fraction was reduced (-7…

Unusual roles of caspase-8 in triple-negative breast cancer cell line MDA-MB-231

Triple-negative breast cancer (TNBC) is a clinically aggressive form of breast cancer that is unresponsive to endocrine agents or trastuzumab. TNBC accounts for ~10-20% of all breast cancer cases and represents the form with the poorest prognosis. Patients with TNBC are at higher risk of early recurrence, mainly in the lungs, brain and soft tissue, therefore, there is an urgent need for new therapies. The present study was carried out in MDA-MB-231 cells, where we assessed the role of caspase-8 (casp-8), a critical effector of death receptors, also involved in non‑apoptotic functions. Analysis of casp-8 mRNA and protein levels indicated that they were up-regulated with respect to the normal…

THE ROLE OF THE EXTRINSIC PATHWAY OF APOPTOSIS IN THE EFFECT INDUCED BY SAHA IN HUMAN HEPATOMA CELLS.

Parthenolide generates reactive oxygen species and autophagy in MDA-MB231 cells. A soluble parthenolide analogue inhibits tumour growth and metastasis in a xenograft model of breast cancer.

Triple-negative breast cancers (TNBCs) are clinically aggressive forms associated with a poor prognosis. We evaluated the cytotoxic effect exerted on triple-negative MDA-MB231 breast cancer cells both by parthenolide and its soluble analogue dimethylamino parthenolide (DMAPT) and explored the underlying molecular mechanism. The drugs induced a dose- and time-dependent decrement in cell viability, which was not prevented by the caspase inhibitor z-VAD-fmk. In particular in the first hours of treatment (1–3 h), parthenolide and DMAPT strongly stimulated reactive oxygen species (ROS) generation. The drugs induced production of superoxide anion by activating NADPH oxidase. ROS generation caused…

Modeling human osteosarcoma in mice through 3AB‐OS cancer stem cell xenografts

Osteosarcoma is the second leading cause of cancer-related death for children and young adults. In this study, we have subcutaneously injected—with and without matrigel—athymic mice (Fox1nu/nu) with human osteosarcoma 3AB-OS pluripotent cancer stem cells (CSCs), which we previously isolated from human osteosarcoma MG63 cells. Engrafted 3AB-OS cells were highly tumorigenic and matrigel greatly accelerated both tumor engraftment and growth rate. 3AB-OS CSC xenografts lacked crucial regulators of beta-catenin levels (E-cadherin, APC, and GSK-3beta), and crucial factors to restrain proliferation, resulting therefore in a strong proliferation potential. During the first weeks of engraftment 3AB-…

Parthenolide sensitizes hepatocellular carcinoma cells to trail by inducing the expression of death receptors through inhibition of STAT3 activation

This article shows that HepG2, Hep3B, and SK-Hep1 cells, three lines of human hepatocellular carcinoma (HCC) cells, are resistant to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Parthenolide, a sesquiterpene lactone found in European feverfew, has been shown to exert both anti-inflammatory and anti-cancer activities. This article demonstrates that co-treatment with parthenolide and TRAIL-induced apoptosis with synergistic interactions in the three lines of HCC cells. In order to explain these effects we ascertained that parthenolide increased either at protein or mRNA level the total content of death receptors TRAIL-R1 and -R2 as well as their surfac…

Janus-Faced role of microRNA let-7d in osteosarcoma 3AB-OS cancer stem cells

Osteosarcoma (OS) is the most common malignancy of bone in children and adolescent. It is a highly invasive and metastatic bone-malignancy because of which, despite therapeutic advances, 30%-50% of patients still die of pulmonary metastasis. As a consequence, there is an urgent need to identify new therapeutic strategies to improve the clinical outcome of the patients. Advances in OS treatment are inconceivable without better understanding of molecular mechanism of osteosarmagenesis and, especially, metastatic processes. Growing evidence suggests that cancer stem cells (CSCs), which have self-renewing and malignant potential, are at the root of tumor growth and relapse. Thus, a challenge fo…

Parthenolide induces caspase-independent and AIF mediated cell death in tumor cells

The cannabinoid WIN induces DR5 receptor expression in hepatoma cells sensitizing them to TRAIL signal

SYNERGISTIC APOPTOTIC INTERACTION BETWEEN THE HDAC INHIBITOR SAHA AND THE PROTEASOME INHIBITOR BORTEZOMIB IN HUMAN HEPATOMA CELLS.

Bortezomib induces in HepG2 cells IkappaBalpha degradation mediated by caspase-8.

The present paper demonstrates that the proteasome inhibitor bortezomib, which behaves as an apoptotic agent in hepatoma HepG2 cells, caused in these cells a decrease in IkappaBalpha level and a consequent increase in NF- kappaB activity. The effect already appeared at 4 h of treatment and preceded the onset of apoptosis which was observed at 24 h. Our results demonstrate that bortezomib-induced IkappaBalpha degradation occurred in conjunction with the activation of caspase-8; moreover, the decrease in IkappaBalpha level was prevented in a dose-dependent manner by the addition of z-IETD, a specific inhibitor of caspase-8. Bortezomib caused the same effects in non-tumor Chang liver cells, wh…

The synthetic cannabinoid WIN sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis by activating p8/CHOP/DR5 axis.

In this paper we demonstrate that the synthetic cannabinoid WIN sensitizes human hepatocellular carcinoma (HCC) cells to apoptosis mediated by TNF-related apoptosis inducing ligand (TRAIL). The apoptotic mechanism induced by treatment with WIN/TRAIL combination involved the loss of the mitochondrial transmembrane potential and led to the activation of caspases. In HCC cells WIN treatment induced up-regulation of TRAIL death receptor DR5, an effect which seemed to be related to the increase in the level of p8 and CHOP, two factors implicated in cellular stress response and apoptosis. This relationship was suggested by the observation that the down-regulation of p8 or CHOP by specific siRNAs …

RB1 in cancer: Different mechanisms of RB1 inactivation and alterations of pRb pathway in tumorigenesis.

Loss of RB1 gene is considered either a causal or an accelerating event in retinoblastoma. A variety of mechanisms inactivates RB1 gene, including intragenic mutations, loss of expression by methylation and chromosomal deletions, with effects which are species-and cell type-specific. RB1 deletion can even lead to aneuploidy thus greatly increasing cancer risk. The RB1gene is part of a larger gene family that includes RBL1 and RBL2, each of the three encoding structurally related proteins indicated as pRb, p107, and p130, respectively. The great interest in these genes and proteins springs from their ability to slow down neoplastic growth. pRb can associate with various proteins by which it …

JNK AND AP-1 MEDIATE APOPTOSIS INDUCED BY BORTEZOMIB IN HEPATOMA HEPG2 CELLS.

L'utilizzo di scarti di vetro nei conglomerati bituminosi per strato di base

Let-7d miRNA Shows Both Antioncogenic and Oncogenic Functions in Osteosarcoma-Derived 3AB-OS Cancer Stem Cells

Osteosarcoma (OS), an aggressive highly invasive and metastatic bone-malignancy, shows therapy resistance and recurrence, two features that likely depend on cancer stem cells (CSCs), which hold both self-renewing and malignant potential. So, effective anticancer therapies against OS should specifically target and destroy CSCs. We previously found that the let-7d microRNA was downregulated in the 3AB-OS-CSCs, derived from the human OS-MG63 cells. Here, we aimed to assess whether let-7d modulation affected tumorigenic and stemness properties of these OS-CSCs. We found that let-7d-overexpression reduced cell proliferation by decreasing CCND2 and E2F2 cell-cycle-activators and increasing p21 an…

The synergistic effect of SAHA and parthenolide in MDA-MB231 breast cancer cells

Abstract: The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagi…

Histone deacetylase inhibitors: apoptotic effects and clinical implications (Review).

It has been shown that epigenetic modifications play an important role in tumorigenesis. Thus, affecting epigenetic tumorigenic alterations can represent a promising strategy for anticancer targeted therapy. Among the key chromatin modifying enzymes which influence gene expression, histone acetyltransferases (HATs) and histone deacetylases (HDACs) have recently attracted interest because of their impact on tumor development and progression. Increased expression of HDACs and disrupted activities of HATs have been found in several tumor types, with a consequent hypoacetylated state of chromatin that can be strictly correlated with low expression of either tumor suppressor or pro-apoptotic gen…

Paclitaxel and beta-lapachone synergistically induce apoptosis in human retinoblastoma Y79 cells by downregulating the levels of phospho-Akt.

Paclitaxel (PTX) and beta-lapachone (LPC) are naturally occurring compounds that have shown a large spectrum of anticancer activity. In this article we show for the first time that PTX/LPC combination induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and LPC (1.5 microM) caused biochemical and morphological signs of apoptosis at 48 h of treatment. These effects were accompanied by potent lowering in inhibitor of apoptosis proteins and by activation of Bid and caspases 3 and 6 with lamin B and PARP breakdown. PTX/LPC combination acted by favoring p53 stabilization through a lowering in p-Akt levels and in ps166-MDM…

Staurosporine-induced apoptosis in Chang liver cells is associated with down-regulation of Bcl-2 and Bcl-XL.

A potent inhibitor of serine/threonine kinases, staurosporine exerts antiproliferative and apoptotic effects in many cancer cells, although the exact mechanism of its action is still unclear. This study examines the effects of staurosporine on Chang liver cells, an immortalized non-tumor cell line, in comparison with those caused in HuH-6 and HepG2 cells, two human hepatoma cell lines. Our results provide evidence that staurosporine promotes apoptosis in Chang liver cells as observed by flow cytometric analysis and acridine orange/ethidium bromide staining. The effect appeared already after 8 h of treatment and increased with treatment time and dose. After 48 h of exposure to 200 nM stauros…

Synergistic effects induced by combinations of the synthetic cannabinoid WIN55,212-2 and the death receptor ligand TRAIL in hepatoma HepG2 cells.

Differentiative pathway induced by 3-aminobenzamide in MG-63 human osteosarcoma cells.

Uridine enhances the cytotoxic effect of D-glucosamine in rat C6 glioma cells.

This paper studies the influence of uridine on the effects exerted by D-glucosamine in rat C6 glioma cells. 2 mM uridine increased markedly both the cytotoxic effect of the aminosugar and the inhibition of thymidine incorporation into acid-insoluble fraction. Furthermore the complete resumption of the capacity to incorporate either 3H-thymidine or 3H-mannose which was observed after the removal of the aminosugar, was impeded when the cells were treated contemporaneously with D-glucosamine and uridine. An exposure for 4 hr to 20 mM glucosamine alone enhanced about 15-fold the cellular pool of UDP-N-acetylhexosamines; the addition of 2 mM uridine intensified the expansion of this pool, which …

Development of nucleoside phosphotransferase activity in the cerebral hemispheres of embryonal and adult chick.

In the cerebral hemispheres of the chick embryo, the level of nucleoside phosphotransferase activity is much higher than that of thymidine kinase and it increases progressively during development up to the adult stage. Therefore nucleoside phosphotransferase is not coupled with DNA synthesis.

Sodium phenylbutyrate induces apoptosis in human retinoblastoma Y79 cells: The effect of combined treatment with the topoisomerase I-inhibitor topotecan

Our results demonstrate that sodium phenylbutyrate, a compound with a low degree of toxicity, exerted a cytotoxic effect on human retinoblastoma Y79 cells in a time- and dose-dependent manner. Treatment of Y79 cells for 72 h with phenylbutyrate reduced cell viability by 63% at 2 mM and 90% at 4 mM. Cell death caused by phenylbutyrate exhibited the typical features of apoptosis, as shown by light and fluorescent microscopy. Western blot analysis demonstrated that exposure of Y79 cells to phenylbutyrate decreased the level of the antiapoptotic factor Bcl-2 and induced the activation of caspase-3, a key enzyme in the execution phase of apoptosis. Moreover, treatment with phenylbutyrate markedl…

Synergistic cytotoxic interaction of the HDAC inhibitor SAHA with the natural compound parthenolide in MDA-MB231 breast cancer cells.

THE APOPTOTIC EFFECT INDUCED IN HepG2 CELLS BY INHIBITORS OF HISTONE DEACETYLASES IS CORRELATED WITH ACETYLATION OF p53 AND HISTONES.

Synergistic effect of the HDAC inhibitor SAHA and the sesquiterpene lactone parthenolide in triple negative breast cancer cells.

Partial purification and some properties of a nucleoside phosphotransferase of chick embryos.

A nucleoside phosphotransferase purified about 40fold from chick embryos utilizes efficiently as phosphate donors deoxyribonucleoside and pyrimidine ribonucleoside monophosphates, whereas the pyrimidine deoxyribonucleoside appear to be the preferred acceptors of phosphate. The enzyme is very unstable to heat, dilution and dialysis. A marked enhancement in the stability is caused by nucleotides and it seems associated with the formation of an aggregated state of the protein.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid sensitizes human hepatocellular carcinoma cells to TRAIL-induced apoptosis by TRAIL-DISC activation

Abstract This paper shows that the histone deacetylase inhibitor SAHA sensitised at sub-toxic doses human hepatocellular carcinoma cells (HepG2, Hep3B and SK-Hep1) to TRAIL-induced apoptosis, while it was ineffective in primary human hepatocytes (PHHs). In particular in HCC cells SAHA increased the expression of death receptor 5 (DR5) and caused a decrement of c-Flip. These two modifications provoked in the presence of TRAIL the rapid production of TRAIL-DISC and the activation of caspase-8. Consequently SAHA/TRAIL combination induced many apoptotic events, such as a cleavage of Bid into tBid, dissipation of mitochondrial membrane potential, activation of caspase-3 with the consequent cleav…

Il partenolide stimola la produzione di ROS e autofagia in cellule di carcinoma mammario MDA-MB231.Il suo analogo solubile DMAPT inibisce la crescita e le metastasi di xenotrapianti di tumori mammari.

Synergistic cytotoxic interactions between sodium butyrate, MG132 and camptothecin in human retinoblastoma Y79 cells.

This paper studies the effects caused in human retinoblastoma Y79 cells by treatment with combinations of sodium butyrate, the inhibitor of topoisomerase I camptothecin and the inhibitor of 26S proteasome MG132. The combination of sodium butyrate and camptothecin resulted in a strong synergistic cytotoxicity, as revealed by combination indices of 0.77 and 0.52 calculated at IC(50) and IC(75). Synergistic interactions were also demonstrated for combinations of sodium butyrate and MG132, camptothecin and MG132 and for a combination of all three compounds. The cytotoxic effects observed after the combined treatments can be considered a consequence of apoptosis, as suggested by the appearance o…

Insulin-like growth factors in chick embryo retina during development.

Evidence exists supporting an important role for insulin-like growth factors (IGFs) during fetal growth. In the present report we performed studies to define whether developing chick retina contains IGFs and whether IGFs play a role in the growth of this tissue. We have shown that both IGF-I and IGF-II are present in chick embryo retina throughout development (7th-18th day). The highest values, when expressed as ng/g of tissue, were found in the youngest retinas studied (7th-9th day) and at 16th-18th day of development. During whole development the content of IGF-II was about two to three times higher than that ascertained for IGF-I. The tissue also contains cell-surface binding for IGFs. H…

Effects of N2, O2'-dibutyril cyclic GMP on the nucleoside phosphotransferase activity of the retina of the chick embryos.

In the retina of the chick embryo, 2 different forms of nucleoside phosphotransferase take part in the phosphorylation of thymidine. One is an unstable form with higher molecular weight. The other with lower m. wt is a stable form. This paper shows that N2, O2′-dibutyril cyclic GMP causes a marked decrement of the activity of the unstable nucleoside phosphotransferase.

Kinetic properties of a nucleoside phosphotransferase of chick embryo

1. A nonspecific nucleoside phosphotransferase (nucleotide : 3'-deoxynucleotide 5'-phosphotransferase, EC 2.7.1.77), purified from chick embryos, catalyzes the transfer of phosphate ester from a nucleotide donor to a nucleoside acceptor. 2. The enzyme exhibits sigmoidal kinetics with respect to nucleoside monophosphate donors, but with respect to nucleoside di- or triphosphate donors and nucleoside acceptors hyperbolic kinetics were obtained. 3. The nucleoside phosphotransferase of chick embryo is unstable to heat and is protected from inactivation by a large number of nucleosides. 4. Nucleoside di- and triphosphates lower both the concentration of nucleoside monophosphates required for hal…

Synergistic interaction between Parthenolide and TRAIL induces apoptosis in human hepatocarcinoma cells.

Partenolide, a natural compound used in traditional medicine for its anti-inflammatory activity, has recently shown anti-tumor and apoptotic effects. Our studies demonstrated that HepG2; Hep3B and SK-Hep1 hepatocarcinoma cells, which are resistant to human recombinant TRAIL, are potently sensitized to TRAIL-induced apoptosis by low doses of parthenolide resulting in a marked synergist effect. To clarify the mechanism that accounts for this interaction, we demonstrated that parthenolide/TRAIL combination markedly increased DR4 and DR5. These effects might be correlated with STAT proteins modifications. In fact parthenolide and parthenolide/TRAIL combination decreased STAT3 and STAT5 and thei…

Parthenolide induces caspase-independent cell death in osteosarcoma, melanoma and breast cancer cells through the induction of oxidative stress.

Parthenolide, a sesquiterpene lactone found in European feverfew, is used in traditional medicine for its anti-inflammatory activity. In addition, parthenolide has been considered as a novel and effective anti-tumor agent because it induces cytotoxic effects in several tumor cell lines. Our studies demonstrated that parthenolide exerted strong cytotoxic effects in osteosarcoma MG63 and melanoma SK-Mel28 cells in culture. Staining with Hoechst 33342 revealed in most cells after brief periods of treatments (3-5h) chromatin condensation and fragmentation, while only few cells were PI-positive. Prolonging the treatment (5-14h) PI-positive cells strongly augmented, denouncing the increase of nec…

Retinoblastoma: History of His Identification, Characterization and Treatment

The first description of a tumor resembling retinoblastoma (RB) was provided on 1597 by Pieter Pauw, who described a malignancy invading the orbit, the temporal region, and the cranium, filled with a "substance similar to brain tissue mixed with thick blood and like crushed stone". Since then, a number of retinal tumors were described and named until the 1922 when Verhoeff called these tumors RB, a term that the American Ophthalmological Society adopted in 1926. In 1971 Knudson focused on RB, and proposed his ‘two-hit’ theory of the molecular etiology of RB. In 1986, the RB1 gene was identified and the ‘two-hit’ theory of Knudson was validated. Successively, new studies in developing retina…

SAHA induces acetylation of p53 and histones in human hepatoma HepG2 cells

Parthenolide induces caspase-independent and AIF-mediated cell death in human osteosarcoma and melanoma cells

The mechanism of the cytotoxic effect exerted by parthenolide on tumor cells is not clearly defined today. This article shows that parthenolide stimulates in human osteosarcoma MG63 and melanoma SK-MEL-28 cells a mechanism of cell death, which is not prevented by z-VAD-fmk and other caspase inhibitors. In particular treatment with parthenolide rapidly stimulated (1-2 h) reactive oxygen species (ROS) generation by inducing activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and NADPH oxidase. This event caused depletion of thiol groups and glutathione, NF-κB inhibition, c-Jun N-terminal kinase (JNK) activation, cell detachment from the matrix, and cellular shrinkage. The increa…

THE APOPTOTIC EFFECT EXERTED IN HUMAN HEPATOMA CELLS BY THE INHIBITOR OF HISTONE DEACETYLASE SAHA EITHER ALONE OR IN COMBINATION WITH BORTEZOMIB.

Histone deacetylase inhibitors in cancer therapy: Biological and clinical implications.

Influencing epigenetic tumorigenic modifications is an exciting strategy for anticancer drug development. Acetylation/deacetylation of histones, a process catalysed by histone acetyltransferases (HATs) and Histone deacetylases (HDACs) respectively, is an important modality to regulate chromatin remodelling and consequent gene expression. Aberrant expression of HDACs and disrupted activities of HATs have been found in several tumor types, implicating their involvement in cancer. Histone deacetylase inhibitors (HDACi) represent a new and promising class of anti-tumor drugs that influence gene expression by enhancing the acetylation of histones. HDAC inhibitors have been shown to have potent a…

Hsp72 controls bortezomib-induced HepG2 cell death via interaction with pro-apoptotic factors.

The proteasome inhibitor bortezomib is an efficacious inducer of apoptosis in the hepatoma HepG2 cell line. This study shows that bortezomib increased in these cells the level of the survival factor Hsp72 in a time- and dose-dependent manner. In a first phase of treatment, Hsp72 rapidly increased so that at 24 h of incubation with 50 nM bortezomib its level was approximately five-fold higher than the control. In this phase Hsp72 seemed to play a role in preventing HepG2 cell death, since it interacted with and sequestered the pro-apoptotic factors p53, AIF, Bax and Apaf-1. During a second day of treatment, although the nuclear levels of Hsp72, p53 and AIF increased, the interaction of Hsp72…

Mutant p53 gain of function can be at the root of dedifferentiation of human osteosarcoma MG63 cells into 3AB-OS cancer stem cells

Osteosarcoma is a highly metastatic tumor affecting adolescents, for which there is no second-line chemotherapy. As suggested for most tumors, its capability to overgrow is probably driven by cancer stem cells (CSCs), and finding new targets to kill CSCs may be critical for improving patient survival. TP53 is the most frequently mutated tumor suppressor gene in cancers and mutant p53 protein (mutp53) can acquire gain of function (GOF) strongly contributing to malignancy. Studies thus far have not shown p53-GOF in osteosarcoma. Here, we investigated TP53 gene status/role in 3AB-OS cells-a highly aggressive CSC line previously selected from human osteosarcoma MG63 cells-to evaluate its involv…

Environmental, functional and economic criteria for comparing “target roundabouts” with one- or two-level roundabout intersections

The article describes a criterion based on functional, environmental and economic aspects for comparing conventional roundabouts with innovative one- or two-level roundabouts. We compared the performances of eight roundabout types, differing in geometric layout, number of lanes and traffic flow regulation from each other, with regard to vehicle delays and CO2, NOx, PM2.5 and PM10 pollutant emissions. Recently-designed roundabouts - target roundabouts and flyover roundabouts - have also been studied for their undoubted practical interest. By means of closed-form capacity models and CORINAIR methodology, several traffic simulations were carried out to examine a typical annual traffic demand c…

The involvement of the c-Jun/JNK/AP-1 pathway and HSPs in apoptosis induced by the proteasome inhibitor PS-341 (Velcade) in human hepatoma cells.

Degradation of IkBalfa during apoptosis induced by proteasome inhibitors in hepatoma cells.

Suppressive role exerted by microRNA-29b-1-5p in triple negative breast cancer through SPIN1 regulation

MiR-29 family dysregulation occurs in various cancers including breast cancers. We investigated miR-29b-1 functional role in human triple negative breast cancer (TNBC) the most aggressive breast cancer subtype. We found that miR-29b-1-5p was downregulated in human TNBC tissues and cell lines. To assess whether miR- 29b-1-5p correlated with TNBC regenerative potential, we evaluated cancer stem cell enrichment in our TNBC cell lines, and found that only MDA-MB-231 and BT-20 produced primary, secondary and tertiary mammospheres, which were progressively enriched in OCT4, NANOG and SOX2 stemness genes. MiR-29b-1-5p expression inversely correlated with mammosphere stemness potential, and miR-29b…

The apoptotic effects of cisplatin and carboplatin in retinoblastoma Y79 cells.

This study demonstrated that cisplatin and carboplatin stimulate apoptosis in human retinoblastoma Y79 cells, cisplatin being the most effective compound. The apoptotic effect appeared after 8 h and then increased in a time-dependent manner. Treatment with cisplatin and carboplatin also provoked an increase in the level of p53 and p21, and a lowering in Bcl-2. The prolonged exposure of Y79 cells to cisplatin induced resistance to cisplatin, carboplatin and etoposide. The basal level of p53 was in resistant cells higher than in untreated cells, while Bcl-2 was not modified. p53 and Bcl-2 levels did not change after treating of resistant cells with cisplatin, carboplatin or etoposide. However…

PROPRIETÀ STRUTTURALI DI CONGLOMERATI BITUMINOSI MIGLIORATI CON PLASTICHE POST-USO

The role of oxidative stress in apoptosis induced by the histone deacetylase inhibitor suberoylanilide hydroxamic acid in human colon adenocarcinoma HT-29 cells.

Histone deacetylase inhibitors (HDACIs) activate genes that promote cell cycle arrest and apoptosis in a number of tumor cells. This study showed that suberoylanilide hydroxamic acid (SAHA), a potent and commonly used HDACI, induced apoptosis in human colon adenocarcinoma HT-29 cells in a time- and dose-dependent manner. This effect was accompanied by the induction of oxidative stress, dissipation of mitochondrial transmembrane potential and activation of executioner caspases. Moreover, SAHA increased the levels of phosphorylated active forms of p38 and JNK. The addition of either the antioxidant N-acetylcysteine or the specific inhibitor of NADPH oxidase diphenylene iodonium chloride reduc…

Sodium butyrate induces apoptosis in human hepatoma cells by a mitochondria/caspase pathway, associated with degradation of beta-catenin, pRb and Bcl-XL.

Butyrate can promote programmed cell death in a number of tumour cells in vitro. This paper provides evidence that butyrate induces apoptosis in human hepatoma HuH-6 and HepG2 cells but is ineffective in Chang liver cells, an immortalised non-tumour cell line. In both HuH-6 and HepG2 cells, apoptosis appeared after a lag period of approximately 16 h and increased rapidly during the second day of treatment. In particular, the effect was stronger in HuH-6 cells, which were, therefore, chosen for ascertaining the mechanism of butyrate action. In HuH-6 cells, beta-catenin seemed to exert an important protective role against apoptosis, since pretreatment with beta-catenin antisense ODN reduced t…

The purification and properties of nucleoside phosphotransferase from mucosa of chicken intestine

Abstract (1) Nucleoside phosphotransferase (nucleotide:3′-deoxynucleoside 5′-phosphotransferase, EC 2.7.1.77) has been purified from chicken intestine mucosa to apparent homogeneity. The enzyme is represented by a multisubunit protein at different degrees of association. It can dissociate into a compoenent with a marked fall in catalytic activity. (2) The associated forms are similar to the enzyme previously purified from chick embryo as regards: substrate specificity both with respect to nucleoside monophosphate donors and to deoxyribonucleoside acceptors; sigmoidicity in the rate curve with a variable phosphate donor; instability to heat, dilution and lowering of pH; the activating and pr…

COINVOLGIMENTO DELLO STRESS DEL RETICOLO E DEL PROCESSO AUTOFAGICO NELL’APOPTOSI INDOTTA DAL CANNABINOIDE SINTETICO WIN IN CELLULE DI EPATOMA UMANO IN COLTURA

Studi da noi condotti precedentemente hanno dimostrato la capacità del cannabinoide sintetico WIN di indurre apoptosi in cellule di epatocarcinoma umano HepG2 attraverso un meccanismo, dipendente dal fattore trascrizionale PPARg, che prevede riduzione dei livelli di alcuni fattori di sopravvivenza e attivazione di fattori pro-apoptotici della famiglia Bcl-2 (M. Giuliano et al. Biochimie. 2009). Recentemente è, inoltre, emerso che in cellule di glioma i cannabinoidi possono stimolare l’apoptosi attraverso induzione di stress del reticolo endoplasmatico seguito da autofagia. Scopo L'obiettivo del presente studio è stato quello di valutare il coinvolgimento dell’autofagia nel percorso di morte…

SAHA/TRAIL combination induces detachment and anoikis of MDA-MB231 and MCF-7 breast cancer cells

Abstract SAHA, an inhibitor of histone deacetylase activity, has been shown to sensitize tumor cells to apoptosis induced by TRAIL, a member of TNF-family. In this paper we investigated the effect of SAHA/TRAIL combination in two breast cancer cell lines, the ERα−positive MCF-7 and the ERα−negative MDA-MB231. Treatment of MDA-MB231 and MCF-7 cells with SAHA in combination with TRAIL caused detachment of cells followed by anoikis, a form of apoptosis which occurs after cell detachment, while treatment with SAHA or TRAIL alone did not produce these effects. The effects were more evident in MDA-MB231 cells, which were chosen for ascertaining the mechanism of SAHA/TRAIL action. Our results show…

3AB-OS, a new human pluripotent cancer stem cell: properties and expectations for practical applications.

Parthenolide and DMAPT exert cytotoxic effects on breast cancer stem-like cells by inducing oxidative stress, mitochondrial dysfunction and necrosis

Triple-negative breast cancers (TNBCs) are aggressive forms of breast carcinoma associated with a high rate of recidivism. In this paper, we report the production of mammospheres from three lines of TNBC cells and demonstrate that both parthenolide (PN) and its soluble analog dimethylaminoparthenolide (DMAPT) suppressed this production and induced cytotoxic effects in breast cancer stem-like cells, derived from dissociation of mammospheres. In particular, the drugs exerted a remarkable inhibitory effect on viability of stem-like cells. Such an effect was suppressed by N-acetylcysteine, suggesting a role of reactive oxygen species (ROS) generation in the cytotoxic effect. Instead z-VAD, a ge…

HISTONE DEACETYLASE INHIBITORS SENSITIZE HEPATOMA CELLS TO TRAIL_INDUCED APOPTOSIS

The role of cMET in non-small cell lung cancer resistant to EGFR-Inhibitors: Did we really find the target?

Abstract: The advent of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) represented the most important innovation in NSCLC treatment over the last years. However, despite a great initial activity, secondary mutations in the same target, or different alterations in other molecular pathways, inevitably occur, leading to the emergence of acquired resistance, in median within the first year of treatment. In this scenario, the mesenchymal-epidermal transition (cMET) tyrosine kinase receptor and its natural ligand, the hepatocyte growth factor (HGF), seem to play an important role. Indeed either the overexpression or the amplification of cMET, as well as the overexpr…

Anandamide-induced apoptosis in Chang liver cells involves ceramide and JNK/AP-1 pathway

In the present study we demonstrate that anandamide, the most important endogenous cannabinoid, markedly induced apoptosis in Chang liver cells, an immortalized non-tumor cell line derived from normal liver tissue, while it induced only modest effects in a number of hepatoma cell lines. The apoptotic effect was reduced by methyl-beta-cyclodextrin, a membrane cholesterol depletor, suggesting an interaction between anandamide and the membrane microdomains named lipid rafts. Anandamide effects were mediated by the production of ceramide, as demonstrated by experiments performed with the sphingomyelinase inhibitor, desipramine, or with the sphingomyelinase activator, melittin. This conclusion w…

Low doses of paclitaxel potently induce apoptosis in human retinoblastoma Y79 cells by up-regulating E2F1.

Paclitaxel (PTX) is an anticancer drug currently in phase II clinical trials. This study shows for the first time that low doses of PTX (5 nM) potently induce apoptosis in human retinoblastoma Y79 cells. The effect of PTX is accompanied by a potent induction of E2F1 which appears to play a critical role in the effects induced by PTX. PTX induced a dose- and time-dependent effect, with G2/M arrest, cyclines A, E and B1 accumulation and a marked modification in the status of Cdc2-cyclin B1 complex, the major player of the G2/M checkpoint. Apoptosis followed G2/M arrest. An early and prolonged increase in p53 expression with its stabilization by phosphorylation and acetylation and its nuclear …

WIN55,212-2, a synthetic agonist of cannabinoid receptors, sensitizes tumor cells to TRAIL-induced apoptosis.

A short story of 3AB-OS Cancer Stem Cells, a possible model for studying cancer stemness

All tumors contain a population of Cancer Stem Cells (CSCs) responsible for the initiation, growth and development of the tumor and a challenge in cancer research is their identification and eradication. In our laboratory, by chemical treatment of the human osteosarcoma MG63 cell line, we have isolated and characterized the human OS CSC line (3AB-OS). 3AB-OS CSCs have a significant chromosomal complexity and a large number of molecular abnormalities which appear to be strongly congruent with those described in a large number of pediatric and adult osteosarcomas. 3AB-OS cells transdifferentiated in vitro into cells of all three primary germ layers and, when xenografted in athymic mice they w…

THE CANNABINOID AGONIST WIN55,212-2 INDUCES APOPTOSIS IN HUMAN HEPATOMA CELLS.

Nucleoside phosphotransferase in animal tissues. Tissue distribution and kinetic properties

Amphibian, avian and mammal tissues contain a nucleoside phosphotransferase clearly different from those previously described in vegetables and bacteria. Whatever the animal source, the enzyme showed many similar characteristics as far as substrate specificity, dependence upon Mg2+, instability at 37 degrees C, and the protecting effect of nucleotides were concerned. Moreover, when submitted to gel filtration, the enzyme behaved in all cases as a dissociable high molecular weight protein, whose degree of association was controlled by nucleotides. In amphibian and avian tissues multiple forms of the enzyme seem to be present which differ for the substrate concentration at half-maximal veloci…

Induction of apoptosis in human osteosarcoma Saos-2 cells by the proteasome inhibitor MG132 and the protective effect of pRb

Induction of apoptosis in human osteosarcoma Saos-2 cells by the proteasome inhibitor MG132 and the protective effect of pRb

Pattern of polyamines and related monoacetyl-derivatives in chick embryo retina during development

Il partenolide sensibilizza le cellule di epatocarcinoma all'apoptosi indotta da TRAIL

In human retinoblastoma Y79 cells okadaic acid-parthenolide co-treatment induces synergistic apoptotic effects, with PTEN as a key player.

Retinoblastoma is the most common intraocular malignancy of childhood. In developing countries, treatment is limited, long-term survival rates are low and current chemotherapy causes significant morbidity to pediatric patients and significantly limits dosing. Therefore there is an urgent need to identify new therapeutic strategies to improve the clinical outcome of patients with retinoblastoma. here, we investigated the effects of two natural compounds okadaic acid (OKa) and parthenolide (PN) on human retinoblastoma Y79 cells. For the first time we showed that OKa/PN combination at subtoxic doses induces potent synergistic apoptotic effects accompanied by lowering in p-akt levels, increasin…

Biochemical Aspects of Chick Embryo Retina Development: The Effects of Glucocorticoids

In chick embryo retina during development, DNA synthesis and the activities of DNA polymerase, thymidine kinase, thymidylate synthetase, and ornithine decarboxylase (ODC) declined in parallel from day 7 to 12. The administration in ovo of hydrocortisone reduced significantly, particularly at 8-10 days of incubation, both DNA synthesis and the four enzyme activities tested. The effect was dose dependent, reaching the maximum with 50-100 nmol of hydrocortisone, 8-16 h after treatment. The highest inhibition was found for ODC activity (70%), followed by thymidine kinase activity (62%) and DNA synthesis (45%), whereas activities of DNA polymerase and thymidylate synthetase were reduced only by …

APOPTOTIC EFFECTS INDUCED BY ANANDAMIDE IN HEPATIC CELLS.

Parthenolide induces superoxide anion production by stimulating EGF receptor in MDA-MB-231 breast cancer cells

The sesquiterpene lactone parthenolide (PN) has recently attracted considerable attention because of its anti-microbial, anti-inflammatory and anticancer effects. However, the mechanism of its cytotoxic action on tumor cells remains scarcely defined. We recently provided evidence that the effect exerted by PN in MDA-MB-231 breast cancer cells was mediated by the production of reactive oxygen species (ROS). The present study shows that PN promoted the phosphorylation of EGF receptor (phospho-EGFR) at Tyr1173, an event which was observed already at 1  h of incubation with 25  µM PN and reached a peak at 8-16  h. This effect seemed to be a consequence of ROS production, because N-acetylcystein…

The Synthetic Cannabinoid WIN 55,212-2 Sensitizes Hepatocellular Carcinoma Cells to Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Apoptosis by Activating p8/CCAAT/Enhancer Binding Protein Homologous Protein (CHOP)/Death Receptor 5 (DR5) Axis

In this article, we demonstrate that the synthetic cannabinoid R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)-(1-naphthalenyl) methanone mesylate (WIN 55,212-2) sensitizes human hepatocellular carcinoma (HCC) cells to apoptosis mediated by tumor necrosis-related apoptosis inducing ligand (TRAIL). The apoptotic mechanism induced by treatment with WIN/TRAIL combination involved the loss of the mitochondrial transmembrane potential and led to the activation of caspases. In HCC cells, WIN treatment induced the up-regulation of TRAIL death receptor DR5, an effect that seemed to be related to the increase in the level of p8 and CHOP, two factors implicat…

The inhibitory effect of D-glucosamine on thymidine kinase in chick embryo retinas and HeLa cells

D-Glucosamine markedly inhibits thymidine incorporation into the TCA-insoluble fraction and thymidine kinase activity in HeLa cells. Both the inhibitory effects are also observed in isolated retinas of chick embryos. In this case the inhibitory effects are age-dependent and the magnitude of the responses decreases with embryonic development. In addition the time of exposure to D-glucosamine which is necessary to reveal the inhibitory effect on thymidine kinase increases with the age of the embryos.

Okadaic acid-Parthenolide combination at subtoxic doses induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells by upregulating PTEN.

Retinoblastoma is the most common intraocular malignancy afflicting children. The incidence is higher in developing countries, where treatment is limited and long-term survival rates are low. Vincristine, etoposide, and carboplatin -the agents commonly used in the treatment of retinoblastoma- determine side effects causing significant morbidity to pediatric patients and significantly limiting dosing. Thus, identifying new drugs and molecular targets to facilitate the development of novel therapeutics, and finding natural drug combinations to kill cancer cells by synergistically acting at subtoxic doses, may be a good goal. Here, we investigated the effects of two natural compounds, okadaic …

The oxygen radicals involved in the toxicity induced by parthenolide in MDA-MB-231 cells

It has been shown that the sesquiterpene lactone parthenolide lowers the viability of MDA-MB-231 breast cancer cells, in correlation with oxidative stress. The present report examined the different radical species produced during parthenolide treatment and their possible role in the toxicity caused by the drug. Time course experiments showed that in the first phase of treatment (0-8 h), and in particular in the first 3 h, parthenolide induced dichlorofluorescein (DCF) signal in a large percentage of cells, while dihydroethidium (DHE) signal was not stimulated. Since the effect on DCF signal was suppressed by apocynin and diphenyleneiodonium (DPI), two inhibitors of NADPH oxidase (NOX), we s…

The HDAC inhibitor SAHA synergistically stimulates the cytotoxic effect induced by Parthenolide in MDA-MB231 cells

We showed that the sesquiterpene lactone Parthenolide (PN) exerts strong cytotoxic effects on triple negative breast cancer MDA-MB231 cells. Our recent results suggest that PN exerts in these cells a cytoprotective effect, which is due to the activation of mTOR pathway. To inhibit this protective response we employ the HDAC inhibitor SAHA, which is known to prevent AKT/mTOR pathway. We show that PN activates Akt, mTOR, p70S6kinase and NRF2 while SAHA abolishes these effects. Further cell pretreatment with SAHA synergistically sensitizes the cells to the cytotoxic effect of PN. Moreover SAHA alone activates the autophagic process. The addition of PN to SAHA reduces this effect and induces ap…

SAHA induces apoptosis in hepatoma cells and synergistically interacts with the proteasome inhibitor Bortezomib.

Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. This paper shows that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) stimulated at 5-10 microM apoptosis in human hepatoma HepG2 and Huh6 cells, but was ineffective in primary human hepatocytes (PHH). In HepG2 cells SAHA induced the extrinsic apoptotic pathway, increasing the expression of both FasL and FasL receptor and causing the activation of caspase-8. Moreover, SAHA enhanced the level of Bim proteins, stimulated alternative splicing of the Bcl-X transcript with the expression of the proapoptotic Bcl-Xs isoform, induced degradation of Bid into the apoptotic factor t-Bid and dephosphorylat…

Identification of Insulin in Chick Embryo Retina During Development and Its Inhibitory Effect on DNA Synthesis

Incubation of chick embryo retinal explants with insulin resulted in a pronounced inhibition of thymidine uptake and incorporation into trichloroacetic acid-insoluble fraction. The inhibitory effect was highest with explants from embryos at day 7 and day 8, and thereafter it declined markedly with the age of embryos until day 11. A time-course study of the effect revealed that the inhibition occurred after a lag time; both thymidine uptake and incorporation were not altered significantly after 2-6 h of incubation with insulin, but began to decrease thereafter, reaching the maximum after 16 h. The effect was also dose dependent. After 16 h of incubation, the maximal inhibition (65%) was foun…

The transcription factor CHOP is critical for WIN-mediated DR5 up-regulation in apoptosis induced by WIN/TRAIL co-treatment in hepatoma cells

Influence of hydrocortisone on chick embryo retina development

Treatment of chick embryos in ovo with hydrocortisone-21-phosphate (a single dose of 150 micrograms) caused a marked reduction of retinal thymidine kinase activity 24 h later. The inhibitory effect was highest (65-70%) in 8-10-day-old embryos and declined with age, disappearing after day 15. It was accompanied by a reduction in thickness of the retinal layers. Adrenocorticotropic hormone (ACTH) treatment (10 micrograms daily for 2 days) also produced an age-dependent inhibitory effect on retinal thymidine kinase, whereas treatment with a single dose of 200 micrograms of metopirone, a compound that prevents the 11 beta-hydroxylation of steroid molecules in the adrenal glands, impeded the dec…

Pattern of polyamines and related monoacetyl derivatives in chick embryo retina during development.

Polyamines and related monoacetyl derivatives were studied in chick embryo retina during development (6th-19th day). Putrescine, which is high in the first phase of retinogenesis, is necessary to sustain both tissue proliferation and via N-acetylputrescine, gamma-aminobutyric acid synthesis. A later increase in spermidine and particularly spermine may play a role in the last phase of development when the retina reaches maturation. The presence of N1-acetylspermidine already at the 8th day indicates that in chick embryo retina, putrescine synthesis can depend on two separate pathways. The first involves ornithine decarboxylase activity; the second, spermidine/spermine N1-acetyltransferase an…

A loop involving NRF2, miR‐29b‐1‐5p and AKT, regulates cell fate of MDA‐MB‐231 triple‐negative breast cancer cells

The present study shows that nuclear factor erythroid 2-related factor 2 (NRF2) and miR-29b-1-5p are two opposite forces which could regulate the fate of MDA-MB-231 cells, the most studied triple-negative breast cancer (TNBC) cell line. We show that NRF2 activation stimulates cell growth and markedly reduces reactive oxygen species (ROS) generation, whereas miR-29b-1-5p overexpression increases ROS generation and reduces cell proliferation. Moreover, NRF2 downregulates miR-29b-1-5p expression, whereas miR-29b-1-5p overexpression decreases p-AKT and p-NRF2. Furthermore, miR-29b-1-5p overexpression induces both inhibition of DNA N-methyltransferases (DNMT1, DNMT3A, and DNMT3B) expression and …

Synergistic apoptotic effect in HepG2 cells between bortezomib and hystone deacetylase inhibitors

A factor derived from chick embryo retina which inhibits DNA synthesis of retina itself.

Chick embryo retinas contain a peptide factor that inhibits DNA synthesis in explants of chick embryo retina. The inhibitory factor, obtained by acid/ethanol extraction from 15-day-old chick embryo retinas, was partially purified by affinity chromatography on heparin-sepharose CL-6B and gel filtration on Sephadex G-100. The inhibitor reduced DNA synthesis with maximal effects observed in retinal explants from 7 to 8-day-old chick embryos. The inhibitory effect became apparent after 10 h of incubation and reached the maximum levels after 16 h. DNA-inhibiting activity was heat and acid-stable and was destroyed by trypsin and alkaline treatments. The inhibitory effect was observed in retinal e…