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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 that SAHA decreased the level of c-FLIP, thus favouring the interaction of TRAIL with the specific death receptors DR4 and DR5 and the consequent activation of caspase-8. These effects increased when the cells were treated with SAHA/TRAIL combination. Because z-IEDT-fmk, an inhibitor of caspase-8, prevented both the cleavage of the focal adhesion-kinase FAK and cell detachment, we suggest that activation of caspase-8 can be responsible for both the decrement of FAK and the consequent cell detachment. In addition, treatment with SAHA/TRAIL combination caused dissipation of ΔΨ(m), activation of caspase-3 and decrement of both phospho-EGFR and phospho-ERK1/2, a kinase which is involved in the phosphorylation of BimEL. Therefore, co-treatment also induced decrement of phospho-BimEL and a concomitant increase in the dephosphorylated form of BimEL, which plays an important role in the induction of anoikis. Our findings suggest the potential application of SAHA in combination with TRAIL in clinical trials for breast cancer.
Biochimie 07/2011; 94(2):287-99. · 3.02 Impact Factor
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ABSTRACT: The importance of electrofunctional examinations (electroretinography, electro-oculography and visual evoked potentials) in orbital diseases is emphasized. Although such tests cannot give the same support to the clinical diagnosis as ultrasonography or CT scanning, they do give information about the functional state of the various orbital components. Visual evoked potentials can monitor the functionality of the optic nerve during and after trauma or compressive orbital diseases; electroretinography shows retinal changes secondary to traumatic or vascular orbital diseases, while electro-oculography allows to record extraocular muscle dysfunction.
07/2009; 2(3):161-164.
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ABSTRACT: 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 immunocomplexes, obtained from nuclear extracts using an antibody against p53, revealed the presence of acetylated p53 together with acetylated forms of histones and histone acetyltransferases p300 and PCAF. Experiments performed using pifithrin-alpha, a reversible inhibitor of p53, showed a correlation between acetylation of p53 and induction of apoptosis. In addition treatment with siRNA against p53 indicated that p53 is involved in the acetylation of histones. In conclusion, this report suggests that complexes constituted by acetylated p53, acetylated histones and coactivators can play a central role in HDACI-induced apoptosis in HepG2 cells.
International Journal of Oncology 02/2008; 32(1):177-84. · 2.40 Impact Factor
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ABSTRACT: 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 dephosphorylation and inactivation of the anti-apoptotic factor Akt. Consequently, SAHA caused loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, activation of caspase-3 and degradation of PARP. Interestingly, a combination of suboptimal doses of SAHA (1 microM) and bortezomib (5-10 nM), a potent inhibitor of 26S proteasome, synergistically induced apoptosis in both HepG2 and Huh6 cells, but was ineffective in PHH. Combined treatment increased with synergistic effects the expression levels of c-Jun, phospho-c-Jun and FasL and the production of Bcl-Xs. These effects were accompanied by activation of Bid, caspase-8 and 3. In conclusion, SAHA stimulated apoptosis in hepatoma cells and exerted a synergistic apoptotic effect when combined with bortezomib. In contrast, these treatments were quite ineffective in inducing apoptosis in PHH. Thus, our results suggest the potential application of the SAHA/bortezomib combination in clinical trials for liver cancer.
APOPTOSIS 08/2007; 12(7):1327-38. · 4.79 Impact Factor
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ABSTRACT: Histone deacetylase (HDAC) inhibitors represent a promising group of anticancer agents. This paper shows that the HDAC inhibitor
suberoylanilide hydroxamic acid (SAHA) stimulated at 5–10μM 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 dephosphorylation and inactivation of the anti-apoptotic factor Akt. Consequently,
SAHA caused loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, activation of caspase-3 and degradation of PARP.
Interestingly, a combination of suboptimal doses of SAHA (1μM) and bortezomib (5–10nM), a potent inhibitor of 26S proteasome,
synergistically induced apoptosis in both HepG2 and Huh6 cells, but was ineffective in PHH. Combined treatment increased with
synergistic effects the expression levels of c-Jun, phospho-c-Jun and FasL and the production of Bcl-Xs. These effects were
accompanied by activation of Bid, caspase-8 and 3.
In conclusion, SAHA stimulated apoptosis in hepatoma cells and exerted a synergistic apoptotic effect when combined with bortezomib.
In contrast, these treatments were quite ineffective in inducing apoptosis in PHH. Thus, our results suggest the potential
application of the SAHA/bortezomib combination in clinical trials for liver cancer.
APOPTOSIS 06/2007; 12(7):1327-1338. · 4.79 Impact Factor
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ABSTRACT: 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 potential with cytochrome c release and activation of caspase-3. In contrast, Chang liver cells exhibited a very low susceptibility to bortezomib-induced apoptosis, which was accompanied by modest modifications in the expression of apoptotic factors. In HepG2 cells bortezomib markedly increased AP-1 activity and the expression of its transcriptional targets such as c-Jun, FasL, BimEL, which are involved in apoptosis. Moreover, AP-1 induced its own production by increasing c-Jun content in the composition of the same AP-1 complex. In addition, bortezomib caused activation of JNK1, which in turn increased the level of phospho-c-Jun as well as stimulated the activation of caspase-3 and t-Bid, two fundamental apoptotic factors. Interestingly, siRNA silencing of c-Jun or JNK1 reduced HepG2 cell susceptibility to apoptosis and prevented the increase in AP-1 activity. Both JNK-1 and AP-1 thus exerted a crucial role in bortezomib-induced apoptosis. Differently, in Chang liver cells the different composition of AP-1 complex as well as the failure of JNK activation seemed to be responsible for the low susceptibility to apoptosis. Given the high susceptibility of hepatoma cells to bortezomib, our results suggest the potential application of this compound in clinical trials for liver cancers.
APOPTOSIS 05/2006; 11(4):607-25. · 4.79 Impact Factor
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ABSTRACT: 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 potential with cytochrome c release and activation of caspase-3. In contrast, Chang liver cells exhibited a very low susceptibility to bortezomib-induced
apoptosis, which was accompanied by modest modifications in the expression of apoptotic factors.
In HepG2 cells bortezomib markedly increased AP-1 activity and the expression of its transcriptional targets such as c-Jun,
FasL, BimEL, which are involved in apoptosis. Moreover, AP-1 induced its own production by increasing c-Jun content in the
composition of the same AP-1 complex. In addition, bortezomib caused activation of JNK1, which in turn increased the level
of phospho-c-Jun as well as stimulated the activation of caspase-3 and t-Bid, two fundamental apoptotic factors. Interestingly, siRNA silencing of c-Jun or JNK1 reduced HepG2 cell susceptibility
to apoptosis and prevented the increase in AP-1 activity. Both JNK-1 and AP-1 thus exerted a crucial role in bortezomib-induced
apoptosis. Differently, in Chang liver cells the different composition of AP-1 complex as well as the failure of JNK activation
seemed to be responsible for the low susceptibility to apoptosis. Given the high susceptibility of hepatoma cells to bortezomib,
our results suggest the potential application of this compound in clinical trials for liver cancers.
APOPTOSIS 03/2006; 11(4):607-625. · 4.79 Impact Factor
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ABSTRACT: 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 the content of beta-catenin and anticipated the onset of apoptosis at 8 h of exposure to butyrate. Moreover, in HuH-6 cells, butyrate induced loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspase 9 and caspase 3, and degradation of poly(ADP-ribose) polymerase. In addition, during the second day of treatment, beta-catenin, pRb, and cyclins D and E were diminished and the phosphorylated form of pRb disappeared. Also, the content of the anti-apoptotic factor Bcl-XL fell markedly during this period, while that of the pro-apoptotic factor Bcl-Xs increased. These effects were accompanied by an increase in both Bcl-XL and Bcl-Xs mRNA transcripts, as ascertained by reverse transcriptase-polymerase chain reaction. Our results suggest that caspases have a crucial role in butyrate-induced apoptosis. This conclusion is supported by the observation that the inhibitors of caspases, benzyloxy carbonyl-Val-Ala-Asp-fluoromethylketone and benzyloxy carbonyl-Asp-Glu-Val-Asp-fluoromethylketone, prevented apoptosis and the decrease in Bcl-XL, pRb, cyclins and beta-catenin. These effects were most probably responsible for the increased sensitivity of the cells to butyrate-induced apoptosis, which was observed on the second day of treatment.
European Journal of Cancer 07/2004; 40(9):1441-52. · 5.54 Impact Factor
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Cell Death and Differentiation 09/2003; 10(8):930-2. · 8.85 Impact Factor
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ABSTRACT: 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 that HepG2 cells contain a very low level of Bcl-2 and a much higher level of Bcl-XL, another antiapoptotic factor of the same family. When the cells were exposed to MG132 the level of Bcl-XL diminished, while a new band, corresponding to the expression of the proapoptotic protein Bcl-XS was detected. MG132 also caused the release of cytochrome c from mitochondria and the activation of caspase-3 with the consequent degradation of poly-ADP ribose polymerase (PARP). The observation that the broad spectrum caspase inhibitor z-VAD markedly reduced the apoptotic effect of the drug clearly demonstrated that caspases play an important role in MG132-induced apoptosis. MG132 exerted a modest effect on the viability of Chang liver cells which primarily depended on the G2/M arrest of cell cycle while only a small percentage of apoptotic cells was found. The remarkable differences in the effects induced by MG132 in Chang liver cells and HepG2 cells made us hypothesise the potential use of proteasome inhibitors in hepatocarcinoma therapy.
International Journal of Oncology 11/2002; 21(4):857-65. · 2.40 Impact Factor
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ABSTRACT: This 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 with camptothecin caused a threefold increase in the activity of c-Jun N-terminal kinase (JNK) and an eightfold increase in the level of phosphorylated c-Jun. The introduction of the RB gene into Saos-2 cells reduced the rate of cell growth. Moreover, stable clones of transfected cells were resistant to camptothecin. Exposure to 100 nM camptothecin for 72 h reduced the viability of transfected cells by only 10%; moreover, very modest effects were observed on the activity of JNK as well as on the level of phosphorylated c-Jun. The results reported in this paper support the conclusion that the expression of wild-type pRb in Saos-2 cells exerts an anti-apoptotic influence through the control of JNK activity.
FEBS Letters 07/2001; 499(1-2):191-7. · 3.54 Impact Factor
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ABSTRACT: 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 markedly increased the level of acetylated histone-H3. Combined treatment with phenylbutyrate and topotecan, a topoisomerase I-inhibitor, resulted in a clear synergistic effect. We suggest that the effects exerted by phenylbutyrate on Y79 cells essentially depend on modifications of gene expression consequent to histone hyperacetylation.
International Journal of Oncology 07/2001; 18(6):1233-7. · 2.40 Impact Factor
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ABSTRACT: 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 reduced glutathione attenuated arachidonic acid toxicity. Similar findings were obtained using hydroperoxyeicosatetranoic acids (oxygenated metabolites of arachidonic acid which deplete the reduced glutathione pool) and nordihydroguaretic acid, a general inhibitor of lipooxygenase pathway. which may also trigger rapid depletion of reduced glutathione. Melittin, which is known to activate phospholipase A2, also potently induced apoptosis. Arachidonic acid toxicity was inversely related to cell density. This could depend on an increased production of molecules with antiapoptotic effect; insulin-like growth factors could most likely be one of these molecules. These results propose a role for oxidative stress in the cytotoxicity induced by arachidonic acid in Y79 cells and suggest that these cells could be protected from such toxicity as long as sufficient levels of reduced glutathione and survival factors are present.
Experimental Eye Research 05/2000; 70(4):503-17. · 3.26 Impact Factor
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ABSTRACT: This study deals with the apoptotic effect exerted on human retinoblastoma Y79 cells by both sodium butyrate and an inhibitor of 26S proteasome [z-Leu-Leu-Leu-CHO (MG132)] and their synergistic effect. Exposure to sodium butyrate (1-4 mM) induced an accumulation of cells in the G2-M phase that was already visible after 24 h of treatment, when morphological and biochemical signs of apoptosis appeared only in a small number of cells (5-10%). Thereafter, the apoptotic effects increased progressively with slow kinetics, reaching a maximum after 72 h of exposure, when they concerned a large fraction of cells (>75% with 4 mM sodium butyrate). Sodium butyrate stimulated the conversion of procaspase-3 into caspase-3 and also induced the cleavage of poly-(ADP-ribose) polymerase and lamin B, two hallmarks of apoptosis. All of the apoptotic signals were suppressed by benzyloxy carbonyl-Val-Ala-Asp-fluoromethylketone (a general inhibitor of caspase activities), whereas acetyl-Asp-Glu-Val-Asp aldehyde, a specific inhibitor of caspase-3 activity, only induced a partial reversion of the apoptotic effects. Sodium butyrate also decreased the Bcl-2 level, whereas it increased the Bax level and stimulated the release of cytochrome c from the mitochondria, an event that was most likely responsible for the activation of caspase-3. Finally, sodium butyrate activated 26S proteasome, the major extralysosomal degradative machinery, which is responsible for the degradation of short-lived proteins. Consequently, the levels of p53, N-myc, and IkappaBalpha (factors that play regulatory roles in apoptosis) diminished, whereas the nuclear level of nuclear factor kappaB concomitantly increased. Treatment of Y79 cells with MG132 induced apoptosis with more rapid kinetics than with sodium butyrate. The effects appeared after 8 h of incubation, reaching a maximum at 24 h, and they were accompanied by increased levels of N-myc, p53, and IkappaBalpha. MG132 also favored the release of cytochrome c from the mitochondria and increased the activity of caspase-3. When Y79 cells were exposed to combinations of sodium butyrate and MG132, the latter compound suppressed the decreasing effect induced by sodium butyrate on the levels of p53, N-myc, and IkappaBalpha and the increasing effect on the nuclear level of nuclear factor kappaB. Moreover, an increase in the level of Bax and an enhancement in the release of cytochrome c from the mitochondria were observed. Clear synergistic effects concerning the activation of both caspase-3 and apoptosis were induced by a combination of suboptimal doses of sodium butyrate and MG132. The results support the conclusion that MG132 potentiates the apoptotic effect of sodium butyrate by suppressing its stimulatory effect on 26S proteasome activity. Synergistic interactions between butyrate and inhibitors of proteasome could represent a new important tool in tumor therapy and, in particular, the treatment of retinoblastoma.
Cancer Research 11/1999; 59(21):5586-95. · 7.86 Impact Factor
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ABSTRACT: Camptothecin (an inhibitor of topoisomerase I) and etoposide and amsacrine (inhibitors of topoisomerase II) both capable of triggering programmed cell death in Y79 cells, induced a remarkable dose-dependent increase in the level of cyclin E in these cells. Camptothecin was found to be the most effective compound. The effect was not observed when the cells were treated with other inducers of programmed cell death (C2-ceramide, sodium butyrate, interleukin-1beta and tumor necrosis factor), all of which do not damage DNA. The effect, which was completely prevented by inhibitors of macromolecular synthesis, occurred after a lag phase (12 hrs.) and increased concurrently with the rise in programmed cell death (PCD), reaching a maximum after 36 hrs. of incubation, when a large percentage of cells (95%) showed clear PCD signals. We suggest that cyclin E takes part in the final stage of programmed cell death which is induced by topoisomerase inhibitors in Y79 cells.
Cellular and molecular biology 01/1999; 44(8):1229-35. · 0.98 Impact Factor
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ABSTRACT: C2-ceramide, a cell-permeable analogue of ceramide, induced significant, dose- and time-dependent death in human retinoblastoma Y79 cells. Dying cells strongly displayed the morphology of apoptosis as characterized by microscopic evidence of cell shrinkage, membrane blebbing, nuclear and chromatin condensation and degeneration of the nucleus into membrane-bound apoptotic bodies. Upon induction of apoptosis Y79 cells evidence early phosphatidylserine externalization, as shown by annexin V-FITC. Apoptosis was also assessed by monitoring changes in cell granularity by staining with the combined fluorescent dyes acridine orange and ethidium bromide. C2-ceramide induced these morphological changes without a concomitant production of oligonucleosomal fragments responsible for the DNA ladder and without changes in p53 protein level. Apoptosis was accompanied by accumulation of a modified Bcl-2 protein with a slower-mobility form, and by proteolytic cleavage of PARP. The effect seemed to be specific for C2-ceramide, as C2-dihydroceramide, or other amphiphilic lipid analogues, or products of ceramide hydrolysis were ineffective. The effect also depended on mRNA and protein synthesis as it was markedly inhibited by actinomycin D and cycloheximide. Sphingomyelinase and interleukin-1beta, which are known to activate the sphingomyelin turnover leading to ceramide generation, also induced apoptosis mimicking the effects of ceramide. These findings propose ceramide as an activator of the suicidal program in Y79 cells.
Molecular and Cellular Biochemistry 09/1998; 185(1-2):7-15. · 2.06 Impact Factor
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ABSTRACT: 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, camptothecin which is a powerful inducer of apoptosis in sensitive cells, triggered cell death even in resistant cells. Such an effect was not accompanied by any modification in p53 level while Bcl-2 was markedly reduced.
International Journal of Oncology 09/1998; 13(2):225-32. · 2.40 Impact Factor
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ABSTRACT: To examine the apoptotic effect induced in human retinoblastoma Y79 cells by camptothecin, etoposide, and amsacrine, to examine the effect of these drugs on the expression of many apoptosis-related modulators, and to test the antiapoptotic effect exerted by insulin-like growth factor-I (IGF-I).
Morphologic features of apoptosis were demonstrated using acridine orange- ethidium bromide staining and electron microscopy. DNA fragmentation was determined by means of an in situ cell detection procedure (TdT-dUTP terminal nick-end labeling [TUNEL]) or by electrophoresis on agarose gels and was quantified by enzyme-linked immunosorbent assay. The expression of apoptosis-related modulators was studied by western blot analysis. The processing of latent p53 was examined by means of pulse- chase analysis.
Camptothecin, etoposide, and amsacrine induced apoptosis in Y79 cells in a dose-dependent manner; camptothecin was the most efficacious compound. The effect, which was dependent on macromolecular synthesis, appeared after a lag of 8 hours and increased for as long as 24 hours. It was lower in cells treated with IGF-I, a potent mitogenic factor. Camptothecin and etoposide increased the p53 level after 4 hours of treatment, before the onset of apoptosis. This effect seemed to be a consequence of the conversion of latent p53 to one that is transcriptionally active. The drugs also induced an increase in p53-related proteins, such as p21, Bax, and IGF binding protein-3 (IGF-BP3), and caused a significant reduction of the Bcl-2 level. The latter effect was less evident in cells pretreated with IGF-I.
Topoisomerase inhibitors induce apoptosis in Y79 cells. This event is accompanied by a decrease in the expression of Bcl-2, a death antagonist, and an increase in that of Bax, a death agonist. A probable consequence of these modifications is the activation of ICE-like activity with degradation of poly-(adenosine diphosphate [ADP] ribose)-polymerase. Insulin-like growth factor-I exerts an antiapoptotic action in Y79 cells, and this function is most likely reduced by the overexpression of IGF-BP3 that is induced by drug treatment.
Investigative Ophthalmology & Visual Science 08/1998; 39(8):1300-11. · 3.60 Impact Factor
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ABSTRACT: 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 were capable of increasing the level of p53. Among these compounds, Taxol, suramin and sodium butyrate also stimulated the oligonucleosomal fragmentation of DNA, while C2-ceramide, a cell-permeable analogue of ceramide, and vitamin D3 were not effective in the induction of DNA laddering in Y79 cells. Apoptosis was dependent on macromolecular synthesis with all the compounds tested.
Tumor Biology 02/1998; 19(5):356-63. · 1.94 Impact Factor
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ABSTRACT: This paper studies the effect exerted by TGF-beta1 on the development of chick embryo retina cultured in vitro. The addition of TGF-beta1 to retinal explants inhibited DNA synthesis, measured as 3H-thymidine incorporation into trichloroacetic acid-insoluble fraction, while it increased both wet weight and protein content, in particular that of extracellular matrix proteins. Lastly, in explants treated with TGF-beta1 an increment in the level of fibronectin was demonstrated by means of Western blotting analysis.
International Journal of Developmental Neuroscience 01/1998; 15(8):973-81. · 2.42 Impact Factor
