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1.
Eur Rev Med Pharmacol Sci ; 21(6): 1413-1420, 2017 03.
Article in English | MEDLINE | ID: mdl-28387883

ABSTRACT

OBJECTIVE: Candida albicans biofilm is frequently found on artificial surfaces and the infections related to biofilm are difficult to eliminate, as they require the removal of artificial devices and treatment with antifungal drugs. Nowadays, fungal growth in biofilms is difficult to eradicate with conventional antifungal drugs such as fluconazole. Among chelating agents, disodium salt-Ethylene Diamine Tetraacetic Acid (EDTA) is known to have antifungal activity. In this study, we examined the in vitro activity of the EDTA and the antifungal drug fluconazole against C. albicans mature biofilm. MATERIALS AND METHODS: C. albicans ATCC 20191, fluconazole-susceptible strain, was grown at an inoculum starter of 1 x 106 cells/ml for 72 h in 24-well microtiter plates and was further treated for 24 h with EDTA and/or fluconazole. Antifungal activities in biofilms were expressed as reduction in optical density (OD) determined by a 2,3-bis (2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay and compared to untreated biofilms. RESULTS: Colorimetric readings revealed that EDTA alone (at 25 and 2.5 mM) significantly reduced fungal metabolic activity in preformed biofilms. Also, EDTA combined with fluconazole significantly reduced the growth of biofilm when compared to biofilm treated with fluconazole alone (at 25 and 2.5 µg/ml). CONCLUSIONS: Our data suggest that the employment of EDTA or other chemicals destabilizers of the biofilm matrix, in combination with antifungal drugs, could lead to the development of new strategies for the management of infections associated to Candida biofilm. Another relevant result of our study suggests that the initial cell concentration, probably through mechanisms of quorum sensing, affects the cellular viability during the process of biofilm formation.


Subject(s)
Antifungal Agents/pharmacology , Biofilms/drug effects , Candida albicans/drug effects , Edetic Acid/pharmacology , Chelating Agents/pharmacology , Fluconazole/pharmacology , Microbial Sensitivity Tests
2.
Cell Cycle ; 14(7): 1090-102, 2015.
Article in English | MEDLINE | ID: mdl-25564714

ABSTRACT

High mortality among hepatocellular carcinoma (HCC) patients reflects both late diagnosis and low curability, due to pharmacoresistance. Taxol (TAX) is toxic for many human HCC-derived cell lines, yet its clinical efficacy on HCCs is poor. Combining TAX with other drugs appears a promising possibility to overcome such refractoriness. We analyzed whether combining tumor necrosis factor (TNF) with TAX would improve their toxicity. Human HCC-derived cell lines were treated with TAX or TNF, alone or combined. Apoptosis was assessed by morphology and flow-cytometry. Several pro- and anti-apoptotic molecules were evaluated by western blotting and/or enzymatic assay. After a 24 hour treatment, TNF was ineffective and TAX modestly cytotoxic, whereas HCC cells were conditionally sensitized to TNF by TAX. Indeed some relevant parameters were shifted to a prodeath setting: TNF-receptor 1 was increased, SOCS3, c-FLIP and pSTAT3 were markedly downregulated. These observations provide a significant clue to critically improve the drug susceptibility of HCC cells by combining 2 agents, TAX and TNF. The sequential application of TAX at a low dosage followed by TNF for only a short time triggered a strong apoptotic response. Of interest, prior TAX administration could also sensitize to TNF-induced apoptosis in the Yoshida AH-130 hepatoma transplanted in mice. Therefore, scrutinizing the possibility to develop similar combination drug regimens in suitable preclinical models seems highly advisable.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Paclitaxel/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Carcinoma, Hepatocellular/metabolism , Caspases/metabolism , Cell Shape , Cell Survival/drug effects , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Enzyme Activation , Hep G2 Cells , Humans , Liver Neoplasms/metabolism , Male , Mice, Inbred BALB C , Neoplasm Transplantation , Paclitaxel/therapeutic use , Receptors, Tumor Necrosis Factor, Type I/metabolism , STAT3 Transcription Factor/metabolism , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/metabolism , Tumor Necrosis Factor-alpha/therapeutic use
3.
G Ital Med Lav Ergon ; 34(3 Suppl): 79-85, 2012.
Article in Italian | MEDLINE | ID: mdl-23405586

ABSTRACT

The aim of this study is to show the results obtained from measuring energy expenditure (EE) during work, through portable devices, in a group of male construction workers. After defining cardio-respiratory parameters in laboratory, authors applied to all subjects an heart rate monitor for measuring the heart rate (HR) and, at the same time, a calorimeter for measuring energy expenditure (EE). To analyse data obtained, authors calculate the Relative Aerobic Strain (RAS), both for the measurements of EE and for HR detected. Results confirm that in many of the typical activities of construction industry, in particular in those characterised by an higher component of manual engagement compared to foreman, workloads are exceeding limits of the probable threshold fatigue (33% of RAS), both for energy expenditure than for HR measured.


Subject(s)
Construction Industry , Energy Metabolism , Adult , Humans , Male , Middle Aged , Young Adult
4.
Curr Med Chem ; 17(4): 309-20, 2010.
Article in English | MEDLINE | ID: mdl-20015045

ABSTRACT

The biological activity of peroxisome proliferators (PPs) is mediated by a class of receptors, known as PPARs (PP-Activated Receptor), belonging to the nuclear receptor superfamily. Upon ligand binding, PPARs dimerize with retinoid receptors, translocate to the nucleus, recognize specific PP-responsive elements on DNA and transactivate a number of genes. Several processes are regulated by PPARs, such as mitochondrial and peroxisomal fatty acid uptake and beta-oxidation, inflammation, intracellular lipid trafficking, cell proliferation and death. In addition, PPARs have been proposed to act as tumor suppressors or as tumor promoters, depending on the circumstances. In particular, PPs have been extensively studied for their hepatocarcinogenic action in rodents, most often ascribed to their antiapoptotic action. Recent evidence, however, has been provided about the antiproliferative, proapoptotic, and differentiation-promoting activities displayed by PPAR ligands. The present review will focus on the cytotoxic effects exerted by several PPs, among which clofibrate, on different types of tumor cells, with particular reference to the mechanisms of cell death and to their relevance to cancer induction and progression.


Subject(s)
Clofibrate/pharmacology , Cytotoxins/pharmacology , Disease Progression , Neoplasms/pathology , Peroxisome Proliferators/pharmacology , Animals , Clofibrate/adverse effects , Clofibrate/metabolism , Cytotoxins/metabolism , Cytotoxins/toxicity , Humans , Neoplasms/metabolism , Peroxisome Proliferator-Activated Receptors/metabolism , Peroxisome Proliferators/adverse effects , Peroxisome Proliferators/metabolism
5.
Curr Cancer Drug Targets ; 9(5): 608-16, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19508174

ABSTRACT

Muscle wasting, as occurring in cancer cachexia, is primarily characterized by protein hypercatabolism and increased expression of ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1. Myostatin, a member of the TGFbeta superfamily, negatively regulates skeletal muscle mass and we showed that increased myostatin signaling occurs in experimental cancer cachexia. On the other hand, enhanced expression of follistatin, an antagonist of myostatin, by inhibitors of histone deacetylases, such as valproic acid or trichostatin-A, has been shown to increase myogenesis and myofiber size in mdx mice. For this reason, in the present study we evaluated whether valproic acid or trichostatin-A can restore muscle mass in C26 tumor-bearing mice. Tumor growth induces a marked and progressive loss of body and muscle weight, associated with increased expression of myostatin and ubiquitin ligases. Treatment with valproic acid decreases muscle myostatin levels and enhances both follistatin expression and the inactivating phosphorylation of GSK-3beta, while these parameters are not affected by trichostatin-A. Neither agent, however, counteracts muscle atrophy or ubiquitin ligase hyperexpression. Therefore, modulation of the myostatin/follistatin axis in itself does not appear sufficient to correct muscle atrophy in cancer cachexia.


Subject(s)
Cachexia/drug therapy , Follistatin/metabolism , Histone Deacetylase Inhibitors , Hydroxamic Acids/pharmacology , Muscles/drug effects , Muscular Atrophy/metabolism , Myostatin/metabolism , Valproic Acid/pharmacology , Animals , Cachexia/complications , Cachexia/pathology , Colonic Neoplasms/complications , Disease Models, Animal , Drug Evaluation, Preclinical , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta , Hydroxamic Acids/therapeutic use , Mice , Mice, Inbred BALB C , Muscles/metabolism , Muscular Atrophy/complications , Muscular Atrophy/drug therapy , Neoplasm Transplantation , Ubiquitin-Protein Ligases/metabolism , Valproic Acid/therapeutic use
6.
Biochem Pharmacol ; 77(2): 169-76, 2009 Jan 15.
Article in English | MEDLINE | ID: mdl-18983831

ABSTRACT

Peroxisome proliferators (PPs) are a class of compounds that exert their nominal effects through the peroxisome proliferator-activated receptors. PPs, among which clofibrate (CF), have been extensively studied for their hepatocarcinogenic properties in rodents, generally ascribed to their antiapoptotic action. However, previous results demonstrated that various PPs may also have apoptogenic properties. CF, in particular, promptly induces a massive apoptotic death in cell lines established from murine or human hepatomas and from breast or lung cancers as well. The present study was aimed at elucidating the apoptotic pathway(s) triggered by CF in AH-130 cells. The results show that CF-induced cell death is completely blocked by the poly-caspase inhibitor z-VAD-fmk and that caspases 3, 8, and 9 are early activated. Consistently, cytochrome c is released from mitochondria, and CF cytotoxicity is inhibited by cyclosporine A, partially at least. In addition, the occurrence of endoplasmic reticulum (ER) stress is suggested by the observation that the levels of phosphorylated eIF2alpha and JNK increase in CF-treated cells, while the caspase 2 precursor protein levels are concurrently reduced. Finally, some degree of calpain activation also takes place, as suggested by the appearance of fodrin cleavage products. The present findings demonstrate that CF-induced apoptosis in the Yoshida AH-130 cells basically is a caspase-dependent process that involves more than a single mechanisms. Activation of the intrinsic apoptotic pathway and ER stress both play a major and concurrent role, while calpain activation seems to have only a marginal part in the process.


Subject(s)
Apoptosis/drug effects , Clofibrate/pharmacology , Amino Acid Chloromethyl Ketones/pharmacology , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/physiopathology , Caspase 3/metabolism , Caspase 8/metabolism , Caspase 9/metabolism , Cell Death/drug effects , Cell Line, Tumor , Cysteine Proteinase Inhibitors/pharmacology , Humans , Liver Neoplasms/pathology , Liver Neoplasms/physiopathology
7.
Eur J Clin Invest ; 38(7): 531-8, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18578694

ABSTRACT

BACKGROUND/AIMS: Myostatin belongs to the transforming growth factor-beta superfamily and negatively regulates skeletal muscle mass. Its deletion induces muscle overgrowth, while, on the contrary, its overexpression or systemic administration cause muscle atrophy. The present study was aimed at investigating whether muscle depletion as occurring in an experimental model of cancer cachexia, the rat bearing the Yoshida AH-130 hepatoma, is associated with modulations of myostatin signalling and whether the cytokine tumour necrosis factor-alpha may be relevant in this regard. MATERIALS AND METHODS: Protein levels of myostatin, follistatin (myostatin endogenous inhibitor) and the activin receptor type IIB have been evaluated in the gastrocnemius of tumour-bearing rats by Western blotting. Circulating myostatin and follistatin in tumour hosts were evaluated by immunoprecipitation, while the DNA-binding activity of the SMAD transcription factors was determined by electrophoretic-mobility shift assay. RESULTS: In day 4 tumour hosts muscle myostatin levels were comparable to controls, yet follistatin was reduced, and SMAD DNA-binding activity was enhanced. At day 7, both myostatin and follistatin increased in tumour bearers, while SMAD DNA-binding activity was unchanged. To investigate whether tumour necrosis factor-alpha contributed to induce such changes, rats were administered pentoxifylline, an inhibitor of tumour necrosis factor-alpha synthesis that partially corrects muscle depletion in tumour-bearing rats. The drug reduced both myostatin expression and SMAD DNA-binding activity in day 4 tumour hosts and up-regulated follistatin at day 7. CONCLUSIONS: These observations suggest that myostatin pathway should be regarded as a potential therapeutic target in cancer cachexia.


Subject(s)
Cachexia/metabolism , Muscle, Skeletal/metabolism , Muscular Atrophy/metabolism , Signal Transduction/physiology , Transforming Growth Factor beta/metabolism , Analysis of Variance , Animals , Blotting, Western , Cachexia/genetics , Disease Models, Animal , Male , Muscular Atrophy/genetics , Myostatin , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Transforming Growth Factor beta/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
8.
Apoptosis ; 10(4): 777-86, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16133868

ABSTRACT

Rat hepatoma HTC cells are intrinsically resistant to various apoptosis-inducing agents. Strategies to induce death in hepatoma cells are needed and the present experimental study was aimed to investigate the sensitivity of HTC cells to TNF and to clarify the mechanisms of action of this cytokine. Cells were treated with TNF and death mechanisms characterized employing an integration of morphological and biochemical techniques. HTC cells, sensitized to TNF toxicity with cycloheximide, died in a caspase-independent apoptosis-like manner. Although we found no evidence for a direct involvement of lysosomal cathepsins, bafilomycin A1 and ammonium chloride significantly attenuated TNF toxicity. Also desferrioxamine mesylate, an iron chelator, partly protected the cells from TNF, while a complete protection was afforded by combining ammonium chloride and iron chelator. Moreover, HTC were protected from TNF also by lipophylic antioxidants and diphenylene iodonium chloride, a NADPH oxidase inhibitor. These data depict a novel mechanism of TNF-mediated cytotoxicity in HTC cells, in which the endo-lysosomal compartment, NADPH oxidase and an iron-mediated pro-oxidant status contribute in determining a caspase-independent, apoptosis-like cell death.


Subject(s)
Apoptosis/drug effects , Intracellular Space/metabolism , Iron/metabolism , Liver Neoplasms, Experimental/pathology , Tumor Necrosis Factor-alpha/pharmacology , Acids , Animals , Antioxidants/pharmacology , Caspase Inhibitors , Cathepsins/metabolism , Cell Line, Tumor , Cycloheximide/pharmacology , Deferoxamine/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Intracellular Space/drug effects , Liver Neoplasms, Experimental/enzymology , Lysosomes/drug effects , Mitochondria/drug effects , Mitochondria/enzymology , Rats
9.
Int J Oncol ; 26(6): 1663-8, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15870883

ABSTRACT

Cachexia is a syndrome characterized by profound skeletal muscle wasting that frequently complicates malignancies. A number of studies indicate that protein hypercatabolism, largely mediated by classical hormones and cytokines, is the major component of muscle depletion. Impaired regeneration has been suggested to contribute to the reduction of muscle size. In particular, it has been shown that the expression of MyoD, a muscle-specific transcription factor, is down-regulated by cytokines such as TNFalpha and IFNgamma in a NF-kappaB-dependent posttranscriptional manner. The present study investigated whether modulations of the transcription factor MyoD are associated with the onset of muscle wasting in a well established model of cancer cachexia. Rats bearing the Yoshida AH-130 hepatoma develop a condition of muscle protein hypercatabolism, largely dependent on TNFalpha bioactivity. In the gastrocnemius of these animals the expression of MyoD was markedly reduced, paralleling the decrease of muscle weight. This pattern is associated with increased nuclear translocation of AP-1, while DNA-binding assays did not detect any change in NF-kappaB activity. This is the first observation demonstrating that muscle depletion in tumor-bearing rats is associated with a down-regulation of MyoD levels. Although the underlying mechanisms remain to be clarified, this change is compatible with the hypothesis that a reduced expression of molecules involved in the regulation of the regenerative response may concur to muscle wasting in cancer cachexia.


Subject(s)
Muscle, Skeletal/metabolism , MyoD Protein/analysis , Neoplasms, Experimental/metabolism , Wasting Syndrome/etiology , Animals , Cachexia/metabolism , DNA/metabolism , Down-Regulation , Male , Rats , Rats, Wistar , Transcription Factor AP-1/metabolism , Tumor Necrosis Factor-alpha/physiology , Wasting Syndrome/metabolism
10.
Cell Death Differ ; 10(9): 997-1004, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12934074

ABSTRACT

The liver is particularly susceptible to Fas-mediated cytotoxicity. Mice given an adequate parenteral dose of agonistic anti-Fas antibody (aFas) or of FasL are known to develop a devastating liver injury and to die in a few hours. The present work shows that mice lacking TNFR1 and TNFR2 (R(-)) both survive a single dose of aFas, otherwise rapidly lethal, and develop a mild form of hepatic damage, compared to the much more severe liver injury that in a few hours strikes wild-type mice (R(+)), eventually involving increased activity of proteases of different families (caspase 3-, 8-, and 9-like, calpains, cathepsin B). Neither the overall tissue levels of Fas and FasL nor Fas expression at the hepatocyte surface are altered in the liver of R(-) animals. The DNA-binding activity of the NF-kappaB transcription factor is enhanced after aFas treatment, but much more markedly in R(-) than in R(+) mice. Bcl2, while unchanged in untreated animals, is markedly upregulated in R(-) but not in R(+) mice challenged with aFas. The requirement of a normal TNFR1/TNFR2 phenotype for full deployment of the general and liver-specific aFas toxicity in mice most likely implies that treatment with aFas in some way results in activation of the TNFalpha-TNFRs system and that this activation synergizes with Fas-mediated signals in causing the fulminant liver injury and the animal death. The precise cellular and molecular details underlying this interplay between Fas- and TNFRs-mediated signaling systems in the general and liver-specific aFas toxicity largely remain to be clarified.


Subject(s)
Antigens, CD/physiology , Apoptosis , Hepatitis, Animal/etiology , Receptors, Tumor Necrosis Factor/physiology , fas Receptor/metabolism , Animals , Antibodies/toxicity , Antigens, CD/genetics , Fas Ligand Protein , Hepatitis, Animal/metabolism , Hepatitis, Animal/pathology , Liver/pathology , Liver/ultrastructure , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor, Type I , Receptors, Tumor Necrosis Factor, Type II , Tumor Necrosis Factor-alpha/physiology , fas Receptor/immunology
11.
Cytokine ; 19(1): 1-5, 2002 Jul 07.
Article in English | MEDLINE | ID: mdl-12200106

ABSTRACT

The ascites hepatoma Yoshida AH-130 induces loss of body weight and tissue waste. Tumour necrosis factor alpha (TNF-alpha) plays a pivotal role in the pathogenesis of muscle wasting in this model system, but other cytokines, such as interleukin-6, may be involved. In order to verify whether a combined anticytokine treatment may synergistically counteract muscle protein degradation, tumour bearing rats were treated with pentoxyfilline (PTX, an inhibitor of TNF-alpha synthesis), or with suramin (SUR, an antiprotozoal drug blocking the peripheral action of several cytokines including IL-6 and TNF-alpha), or both the drugs, and the effects on muscle proteolytic systems were assessed. Muscle protein loss in the AH-130-bearing rats was associated with increased activity of both the ATP-ubiquitin- and the calpain- dependent proteolytic pathways (246% and 230% of controls, respectively). Both PTX and SUR, either alone or in combination, prevented the depletion of muscle mass and significantly reduced the activity of muscle proteolytic systems. In particular, treatment with SUR, either alone or with PTX, induced a decrease in enzymatic activities to values similar to those of controls. The results obtained in the present paper demonstrate that: (i) muscle depletion in this model is indeed associated with increased proteasome- and calpain-dependent proteolysis, as previously suggested by increased mRNA expression of molecules pertaining to both pathways; (ii) anticytokine treatments effectively reduce muscle protein loss by down-regulating the activity of at least two major proteolitic systems; (iii) SUR is more effective than PTX in reducing the activity of proteolytic systems, possibly because of its multiple anticytokine action.


Subject(s)
Adenosine Triphosphate/metabolism , Calcium/metabolism , Cytokines/antagonists & inhibitors , Ubiquitin/metabolism , Animals , Body Weight/drug effects , Cachexia/prevention & control , Cysteine Endopeptidases/metabolism , Down-Regulation , Interleukin-6/metabolism , Liver Neoplasms/drug therapy , Multienzyme Complexes/metabolism , Muscle, Skeletal/metabolism , Muscles/pathology , Organ Size/drug effects , Proteasome Endopeptidase Complex , Rats , Rats, Wistar , Tumor Necrosis Factor-alpha/metabolism
12.
Anticancer Res ; 22(2B): 1171-6, 2002.
Article in English | MEDLINE | ID: mdl-12168920

ABSTRACT

OBJECTIVE: The authors report their experience in patients with adjuvant systemic 2-interferon with the aim of defining the effectiveness, side-effects, indications and limitations of this treatment. MATERIALS AND METHODS: From January 1989 to December 1996, 123 patients with genital, anorectal and perineal HPV lesions were treated with cryosurgery; adjuvant systemic a2-interferon was administered to 38 of them. There were 76 female and 47 male patients (median age of 29 years, range; 15-56 years). Clinical examinations included: digital rectal examination, head and neck examination, urethral meatus inspection and, in female patients, gynaecological examination; they underwent colposcopylurethroscopy, proctosigmoidoscopy, cystoscopy (in advanced disease); scraping for cytology and PCR analysis, and biopsy for histology. Twenty-three percent of patients had more than one site involved; upper digestive tract involvement was observed in 6.6% and 47% had lesions larger than 6 sqcm. Twenty-five females with genital lesions had esocervical lesions only; ten of them had SIL1, while seven a SIL3. RESULTS: Ninety-eight out of 123 patients (79.7%) were recurrence-free after a median follow-up of 32 months. A recurrence was observed in 25 patients: in univariate analysis, recurrence of disease occurred more frequently in females (p = 0.04), in patients with longer duration of symptoms (p = 0.0002),with wider lesions (p = 0.00015), with head and neck involvement (p < 0.01), and in HIV-positive patients (p = 0.03). In multivariate analysis, duration of symptoms (p = 0.005), head and neck involvement (p = 0.01), and width of lesion > 3 sq cm (p = 0.025) were associated with increased risk CONCLUSION: Our findings confirm the value of cryosurgery in the treatment HPV lesions; it is less traumatic, and gives good aesthetic and functional results; moreover, large lesions may be treated and the depth of cryonecrosis is more suitably adapted. Patients amenable to adjuvant treatment with a2-interferon should have multiorgan involvement, HPV type 16 or 18, lesions >3 sqcm, long lasting symptoms (>6 months) and presence of SIL.


Subject(s)
Anus Neoplasms/therapy , Genital Neoplasms, Female/therapy , Genital Neoplasms, Male/therapy , Interferon-alpha/therapeutic use , Papillomaviridae , Papillomavirus Infections/therapy , Tumor Virus Infections/therapy , Adolescent , Adult , Anus Neoplasms/drug therapy , Anus Neoplasms/surgery , Anus Neoplasms/virology , Chemotherapy, Adjuvant , Cryosurgery , Female , Genital Neoplasms, Female/drug therapy , Genital Neoplasms, Female/surgery , Genital Neoplasms, Female/virology , Genital Neoplasms, Male/drug therapy , Genital Neoplasms, Male/surgery , Genital Neoplasms, Male/virology , Humans , Male , Middle Aged , Papillomavirus Infections/complications , Papillomavirus Infections/drug therapy , Papillomavirus Infections/surgery , Perineum/pathology , Perineum/virology , Tumor Virus Infections/complications , Tumor Virus Infections/drug therapy , Tumor Virus Infections/surgery
13.
Int J Cancer ; 93(2): 179-84, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11410863

ABSTRACT

The expression of different protein kinase C (PKC) isoenzymes has been shown to vary with proliferation rates, differentiation or apoptosis in normal colon crypts. In addition, the activity of some PKC isoenzymes appears to be reduced in colorectal cancer. The aim of the present work was to determine whether modulation of PKC expression would affect the susceptibility of a p53-defective colon carcinoma cell line to different apoptotic treatments. HT-29 cells exhibited sensitivity to paclitaxel (Taxol) and tumor necrosis factor alpha (TNFalpha) in a dose- and time-dependent manner but were relatively resistant to etoposide. Inhibition of PKC activity augmented the susceptibility of HT-29 cells to apoptosis, and phorbol ester induction of PKC reduced such susceptibility. Transfected HT-29(PKC) cells, hyper-expressing the beta1 isoform of PKC, were less sensitive to TNFalpha and paclitaxel than the normal counterpart. The present data 1) indicate that the expression of PKC influences the susceptibility of HT-29 colon cancer cells to apoptotic drugs apparently regardless of their mechanism of action, and 2) suggest paclitaxel as a potential candidate for the treatment of colon cancer, possibly in association with inhibitors of PKC (alpha and beta) at doses not cytotoxic per se.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Isoenzymes/biosynthesis , Protein Kinase C/biosynthesis , Colonic Neoplasms/pathology , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/physiology , HT29 Cells , Humans , Isoenzymes/physiology , Paclitaxel/administration & dosage , Protein Kinase C/physiology , Protein Kinase C beta , Time Factors , Tumor Necrosis Factor-alpha/administration & dosage
14.
Hepatology ; 31(4): 956-65, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10733553

ABSTRACT

Molecular mechanisms of basal and D-amphetamine (AMPH)-induced apoptosis were studied in rat liver nodules, 12 (N12) and 30 (N30) weeks after initiation, and in hepatocellular carcinoma (HCC) induced by diethylnitrosamine in rats subjected to resistant hepatocyte model. Basal apoptosis in hematoxylin/eosin- and propidium iodide-stained sections was higher in nodules and HCC than in normal livers. It sharply increased in all tissues 4 hours after AMPH treatment (10 mg/kg), and declined to basal levels at 8 to 12 hours in liver and N12, but remained high up to 18 hours in N30 and HCC. c-myc, Tgf-alpha, p53, and Bcl-X(S) messenger RNA (mRNA) levels were higher, and Bcl-2 mRNA was lower in N12 and/or N30 and HCC than in normal liver. Four hours after AMPH injection, increase in c-myc and decreases in Bcl-2 and Bcl-X(L) mRNAs occurred in all tissues, whereas p53, Bax, and Bcl-X(S) mRNAs increased in N30 and HCC. These changes disappeared in liver and N12 at 18 hours, but persisted in N30 and HCC. c-Myc, P53, Bcl-2, and Bax proteins in normal liver and HCC +/- AMPH showed similar patterns. Tgf-beta1, Tgf-beta-RIII, CD95, and CD95L mRNA levels underwent slight or no changes in any tissue +/- AMPH. Basal Hsp27 expression was high in nodules and HCC, and was stimulated by AMPH in liver and N12, but not in N30 and HCC. These data suggest a role of dysregulation of Bcl-2 family genes and, at least in atypical lesions, of p53 overexpression, in basal and AMPH-induced apoptosis in nodules and HCCs. Hsp27 does not appear to sufficiently protect atypical lesions against apoptosis.


Subject(s)
Apoptosis/drug effects , Dextroamphetamine/pharmacology , Liver Neoplasms, Experimental/pathology , Precancerous Conditions/pathology , Proto-Oncogene Proteins c-bcl-2/genetics , Animals , Apoptosis/genetics , Coloring Agents , DNA Fragmentation , Diethylnitrosamine , Genes, myc , Genes, p53 , Kinetics , Liver Neoplasms, Experimental/chemically induced , Male , Propidium , Proto-Oncogene Proteins/genetics , RNA, Messenger/metabolism , Rats , Rats, Inbred F344 , bcl-2-Associated X Protein , fas Receptor/genetics
15.
Cancer Detect Prev ; 22(4): 357-66, 1998.
Article in English | MEDLINE | ID: mdl-9674879

ABSTRACT

In the AH-130 hepatoma, a poorly differentiated tumor, maintained by weekly transplantations in rats, a low percentage of cells spontaneously underwent apoptosis, mainly during the transition from logarithmic- to stationary-growth phase. It was possible to induce massive apoptosis of cells by treating them with clofibrate, a peroxisome proliferator and hypolipidemic drug. Similar results were obtained with HepG2 cells. With 1 mM clofibrate, apoptosis began to manifest itself after 1 h of treatment in vitro, and was assessed by morphological analysis, by DNA fragmentation carried out with agarose gel electrophoresis, and with flow cytometric determination of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling. The mechanisms whereby clofibrate induces apoptosis are still unclear. Since the peroxisome proliferator-activated receptor was expressed at a very low level and was not stimulated by clofibrate in the AH-130 hepatoma cells, its involvement seems unlikely. Moreover, lipid peroxidation was not increased after clofibrate treatment. Phospholipids and cholesterol were significantly decreased. The decreased cholesterol content might suggest an inhibition of the mevalonate pathway and, therefore, of isoprenylation of proteins involved in cell proliferation.


Subject(s)
Apoptosis , Clofibrate/pharmacology , Liver Neoplasms, Experimental/pathology , Animals , Apoptosis/genetics , Cell Membrane/drug effects , DNA Fragmentation , DNA Nucleotidylexotransferase , DNA, Neoplasm/analysis , DNA, Neoplasm/drug effects , Deoxyuracil Nucleotides , Flow Cytometry , Hypolipidemic Agents/pharmacology , Liver Neoplasms, Experimental/chemistry , Liver Neoplasms, Experimental/metabolism , Male , Microbodies , Rats , Staining and Labeling
16.
Cell Death Differ ; 4(3): 224-32, 1997 Apr.
Article in English | MEDLINE | ID: mdl-16465232

ABSTRACT

Clofibrate, for a long time in use as a hypolipidemic drug, is a well known peroxisomal proliferator (PP) and hepatocarcinogen in rodents. We show here that in vitro 1 mM clofibrate induces a rapid and massive death of rat AH-130 hepatoma cells. Cell death was prominent already after 4 h of treatment, with a characteristic ;apoptotic' pattern by conventional microscopy. This was further supported by the pronounced chromatin condensation detectable on 4',6-diamine-2'-phenylindole dihydrochloride (DAPI) staining, the clearcut internucleosomal DNA fragmentation on agarose-gel electrophoresis (ladder pattern), and the accumulation of markedly hypochromic cells observed in flow cytometric DNA histograms. Consistently with the apoptotic features of the process, some parameters commonly used to detect cell death, such as plasma membrane permeabilization to trypan blue or propidium iodide, lack of mitochondrial retention of rhodamine 123, or extracellular release of lactate dehydrogenase, were all virtually negative. However, these same parameters became markedly positive after 24 h of treatment, which was suggestive for the occurrence of ;secondary' necrosis among AH-130 cells. By a combination of flow cytometric parameters, after 4 h on 1 mM clofibrate only 41% of the AH-130 cells could still be categorized as viable (i.e., non-apoptotic and non-necrotic), while 46% of cells appeared apoptotic and 13% necrotic. At 24 h, 67% of cells were necrotic, 20% apoptotic and only 13% non-apoptotic and non-necrotic. Apoptosis was also extensive in AH-130 cells treated with another PP such as nafenopin at 1 mM concentration and in human hepatoma HepG2 cells treated with clofibrate. By contrast, clofibrate did not cause apoptosis on primary rat hepatocyte cultures. These observations indicate that: (i) apart from their well-known cell growth-promoting action, PPs such as clofibrate or nafenopin may exert a substantial cytotoxic action on targets such as the AH-130 or HepG2 hepatoma cells; (ii) this cell death evolves from an initial 'apoptotic' to an eventual ;necrotic' pattern; (iii) detection of cell death requires the adoption of a full panel of tests, adequate to cover the whole evolving death pattern, while such tests may even be substantially misleading whenever applied individually; (iv) the cytotoxicity of clofibrate and similar agents on normal and, particularly, tumoural cells may deserve careful reevaluation.

17.
Int J Cancer ; 70(3): 310-4, 1997 Jan 27.
Article in English | MEDLINE | ID: mdl-9033633

ABSTRACT

The kinetics of transport and the processing of procathepsin D (proCD), the precursor of a lysosomal aspartyl protease involved in tumor-cell proliferation and metastasis, were compared in normal and SV-40- or benzo[a]pyrene-transformed 3T3 mouse fibroblasts. Sorting of newly synthesized proCD in normal cells was almost complete within 3 hr, while in transformed cells a fraction of the precursor survives a long time. In both normal and transformed 3T3 cultures, secretion of proCD started at 3 hr of chase. However, in normal cells secretion of proCD remained constant between 3 and 24 hr of chase, while in transformed cells it increased along with the chase incubation. The efficiency of formation of the mannose-6-phosphate group on proCD varied among the 3 cell types, being minimal in benzo[a]pyrene-transformed 3T3 cells. Ammonium chloride, a drug known to disrupt the segregation and to enhance the secretion of lysosomal proenzymes, was 2-fold more effective in normal than in transformed 3T3 cells. Despite vacuolar alkalinization, about one third of proCD was segregated into the endosomal-lysosomal pathway in normal and in transformed 3T3 fibroblasts, indicating the existence in these cells of alternative, mannose-6-phosphate receptor-independent mechanisms for targeting proCD. Thus, while hypersecretion of proCD and reduced sensitivity to vacuolar alkalinization are common features of both transformed cell types, the mechanisms responsible for inefficient segregation of proCD may differ between virally and chemically transformed 3T3 cells.


Subject(s)
3T3 Cells/metabolism , Cathepsin D/metabolism , Enzyme Precursors/metabolism , Mannosephosphates/metabolism , Ammonium Chloride/pharmacology , Animals , Biological Transport/drug effects , Cell Line, Transformed , Mice , Phosphorylation
18.
Biochimie ; 79(12): 717-23, 1997 Dec.
Article in English | MEDLINE | ID: mdl-9523013

ABSTRACT

The T160 protein belongs to the HMG-1 box protein family and preferentially binds to non-B-DNA conformations with no sequence specificity. Its exact role has yet to be defined, though it seems to participate in processes involving DNA, such as replication, transcription and recombination. We have used an antisense RNA strategy to investigate its role in cell growth and proliferation. T160 expression is strongly suppressed by stable introduction of an antisense construct into NIH3T3 cells, and this decrease is accompanied by substantial changes in the growth properties of the stable transfectants. Impaired growth of T160- cells was mainly related to two mechanisms: i) decreased rates of cell proliferation at normal serum concentration; and ii) occurrence of cell death by apoptosis at low serum concentration, as demonstrated by both flow cytometry and microscopy. The finding that decreased T160 availability affects cell proliferation, provides further evidence of its involvement in a basic cell function, such as DNA replication.


Subject(s)
DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , High Mobility Group Proteins/biosynthesis , High Mobility Group Proteins/genetics , RNA, Antisense/pharmacology , 3T3 Cells , Animals , Cell Division/drug effects , Cell Division/genetics , Cell Survival/drug effects , Culture Media, Serum-Free/pharmacology , DNA-Binding Proteins/drug effects , Down-Regulation/drug effects , Down-Regulation/genetics , High Mobility Group Proteins/drug effects , Mice , RNA, Antisense/biosynthesis
20.
Exp Cell Res ; 228(2): 292-305, 1996 Nov 01.
Article in English | MEDLINE | ID: mdl-8912723

ABSTRACT

Exponentially growing L929 cells were continuously exposed to 1 or 10 microM etoposide (VP-16). The effects of such treatment on cell growth, cycle distribution, morphology, and selected biochemical events were examined. DNA synthesis rates were markedly decreased and the protein/DNA ratio increased (unbalanced growth). Growth was blocked, with most cells being cycle arrested by 24 h in (late S-)G2-M. An asynchronous process of cell death then developed. Cells initially shrank into eosinophilic, trypan blue-excluding bodies, which were then released into the medium, and eventually became permeable to trypan blue. Transmission electron microscopy confirmed that dying cells acquired an apoptotic morphotype, with compaction and margination of chromatin, loss of microvilli, and shrinkage of cytoplasm and nucleus. Tissue transglutaminase activity and intensity of immunostaining rapidly increased in treated cultures. Internucleosomal DNA fragmentation could not be detected by agarose gel electrophoresis, yet flow cytometry revealed that the apoptotic bodies had a very low DNA fluorescence (< or = 10% of the 2n value). In agreement with the microscopic findings, this suggested that extensive DNA degradation had occurred in dead cells. While rates of cell loss from the monolayer amounted to 21 and 57% day(-1) (1 and 10 microM VP-16, respectively), apoptotic indexes largely underestimated the extent of the process. These indexes only measured the accumulation of apoptotic bodies, i.e., the balance between their generation and disposal. The latter occurred by mechanisms similar to those that operate in tissues: "secondary necrosis" or phagocytosis by viable homotypic cells in the monolayer ("homophagy").


Subject(s)
Apoptosis/drug effects , Etoposide/pharmacology , Animals , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line , Cell Membrane/drug effects , Cell Membrane/ultrastructure , DNA/analysis , DNA/biosynthesis , DNA/drug effects , Flow Cytometry , Kinetics , Mice , Microscopy, Electron , Microvilli/drug effects , Microvilli/ultrastructure , Time Factors , Transglutaminases/metabolism
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