Effect of glutathione depletion on caspase-3 independent apoptosis pathway induced by curcumin in Jurkat cells.

Curcumin, a yellow pigment from Curcuma longa, exhibits anti-inflammatory, antitumor, and antioxidative properties. Although its precise mode of action has not been elucidated so far, numerous studies have shown that curcumin may induce apoptosis in normal and cancer cells. Previously, we showed that in Jurkat cells curcumin induced nontypical apoptosis-like pathway, which was independent of mitochondria and caspase-3. Now we show that the inhibition of caspase-3 by curcumin, which is accompanied by attenuation of internucleosomal DNA fragmentation, may be due to elevation of glutathione, which increased in curcumin-treated cells to 130% of control. We have demonstrated that glutathione depletion does not itself induce apoptosis in Jurkat cells; though, it can release cytochrome c from mitochondria and caspase-3 from inhibition by curcumin, as shown by Western blot. The level of Bcl-2 protein was not affected by glutathione depletion even upon curcumin treatment. Altogether, our results show that in Jurkat cells curcumin prevents glutathione decrease, thus protecting cells against caspase-3 activation and oligonucleosomal DNA fragmentation. On the other hand, it induces nonclassical apoptosis via a still-unrecognized mechanism, which leads to chromatin degradation and high-molecular-weight DNA fragmentation.

An intervention resembling caloric restriction prolongs life span and retards aging in yeast.

The yeast Saccharomyces cerevisiae has a finite life span that is measured by the number of daughter cells an individual produces. The 20 genes known to determine yeast life span appear to function in more than one pathway, implicating a variety of physiological processes in yeast longevity. Less attention has been focused on environmental effects on yeast aging. We have examined the role that nutritional status plays in determining yeast life span. Reduction of the glucose concentration in the medium led to an increase in life span and to a delay in appearance of an aging phenotype. The increase in life span was the more extensive the lower the glucose levels. Life extension was also elicited by decreasing the amino acids content of the medium. This suggests that it is the decline in calories and not a particular nutrient that is responsible, in striking similarity to the effect on aging of caloric restriction in mammals. The caloric restriction effect did not require the induction of the retrograde response pathway, which signals the functional status of the mitochondrion and determines longevity. Furthermore, deletion of RTG3, a downstream mediator in this pathway, and caloric restriction had an additive effect, resulting in the largest increase (123%) in longevity described thus far in yeast. Thus, retrograde response and caloric restriction operate along distinct pathways in determining yeast longevity. These pathways may be exclusive, at least in part. This provides evidence for multiple mechanisms of metabolic control in yeast aging. Inasmuch as caloric restriction lowers blood glucose levels, this study raises the possibility that reduced glucose alters aging at the cellular level in mammals.

Proliferation and apoptosis of human T cells during replicative senescence--a critical approach.

Normal human T lymphocytes growing in culture undergo replicative senescence. Previously, we have shown that in our conditions polyclonal T cells cease proliferation after about three weeks (Radziszewska et al., 1999, Cell Biol. Int. 23, 97-103). Now we present results of a more detailed analysis of in vitro growth as well as phenotypic changes of T cells. Cell cycle analysis showed that about 20% of cells were in the S phase until the 17th day of culture (young cells). The highest number of mitotic cells (phase G2/M; 10%) was observed during the first week of culture. All not dividing senescent cells were stopped in the G1 phase (after the 30th day of culture). The sub-G1 fraction which represents apoptotic cells did not exceed 8% during the whole period until the 30th day of culture. During in vitro T-cell growth, a rather rapid selection to CD3+ CD8+ cells occurs. In the presenescent (between the 17th and 30th day) and senescent populations the majority of cells (above 90%) were CD8 positive. We also have checked the expression of alpha-chain interleukin-2 (IL-2) receptor (CD25). In young and presenescent cells about one third of cells was CD25 positive, but only 15% in the pool of senescent cells. Immunoblotting analysis of p16 protein recognized previously as a marker of senescent T cells, showed its highest and transient expression in presenescent cells. A critical review of the polyclonal T cell replicative senescence model is presented.

Apoptosis-independent alterations in membrane dynamics induced by curcumin.

Curcumin is a well-known natural compound with antiinflammatory properties. Its antiproliferative effect and ability to modulate apoptotic response are considered essential in cancer therapy. The physicochemical properties of curcumin suggest membranous localization, which prompted an investigation of the mechanisms of membrane disturbances evoked by curcumin. We chose the erythrocyte as a convenient model for studying membrane effects of curcumin and showed its nonspecific, apoptosis-independent way of action. Curcumin was found to expand the cell membrane, inducing echinocytosis. Changes in cell shape were accompanied by transient exposure of phosphatidylserine. Membrane asymmetry was recovered by the action of aminophospholipid translocase, which remained active in the presence of curcumin. Lipids rearrangements and drug partitioning caused changes of lipid fluidity. Such nonspecific effects of curcumin on cellular membranes would produce artifacts of apoptosis measurement, since several methods are based on membrane changes.

Glutathione-independent mechanism of apoptosis inhibition by curcumin in rat thymocytes.

Curcumin (CUR) is a natural yellow dye with antioxidant and scavenging properties present in Curcuma species. It is widely used as an anti-inflammatory, anti-mutagenic and chemopreventive agent. In addition to its inhibitory effect on proliferation, CUR has recently been shown to block dexamethasone-induced programmed cell death (apoptosis) of rat thymocytes. Because cellular thiols seem to play a role in redox regulation of apoptosis, the mechanism of the anti-apoptotic effect of CUR was studied by examining the levels of glutathione and acid-soluble sulfhydryl groups. CUR was shown to prevent the glutathione loss occurring in dexamethasone-treated thymocytes, enhancing intracellular glutathione content at 8 hr to 192% of that of nontreated cells. A 60% increase in acid-soluble sulfhydryl groups was also observed. In the presence of L-buthionine S,R-sulfoximine (BSO, an inhibitor of glutathione synthesis), intracellular glutathione content of thymocytes treated with dexamethasone and CUR fell to 31% and that of the acid-soluble sulfhydryl groups to 23% of control after 8 hr. Unexpectedly, the electrophoretic and flow cytometric studies of DNA fragmentation demonstrated that apoptosis did not occur even after 20 hr of incubation with buthionine S,R-sulfoximine and dexamethasone, while control thymocytes and the cells treated only with buthionine S,R-sulfoximine showed DNA fragmentation at a level corresponding to spontaneous apoptosis. These results show that CUR treatment elevated the concentrations of glutathione and nonprotein sulfhydryl groups, thus preventing their decrease in apoptotic thymocytes. Coadministration of L-buthionine S,R-sulfoximine and CUR did not affect the anti-apoptotic effect of CUR suggesting a glutathione-independent mechanism of cell protection.

Apoptosis-like, reversible changes in plasma membrane asymmetry and permeability, and transient modifications in mitochondrial membrane potential induced by curcumin in rat thymocytes.

Curcumin (diferuoylmethane) is a natural compound with anticarcinogenic activities which is able to exert either proapoptotic or antiapoptotic effects in different cell types. This paper focuses on the sequence and extent of primary events induced by curcumin, in comparison with those occurring during dexamethasone-induced apoptosis in rat thymocytes. It also presents annexin VI-FITC as a new probe for studying membrane asymmetry. Curcumin readily penetrates into the cytoplasm, and is able to accumulate in membranous structures such as plasma membrane, endoplasmic reticulum and nuclear envelope. Curcumin-treated cells exhibit typical features of apoptotic cell death, including shrinkage, transient phosphatidylserine exposure, increased membrane permeability and decrease in mitochondrial membrane potential. However, nuclei morphology, DNA fragmentation, the extent and time-course of membrane changes are different from those observed during dexamethasone-induced apoptosis, suggesting that, despite many similarities, the mode of action and the events triggered by curcumin are different from those occurring during typical apoptosis.

[Two-dimensional electrophoresis of proteins from breast cancer cells MCF-7. Changes in synthesis induced by FGF-2]. / Analyse par electrophorèse bidimensionnelle des protéines de cellules de cancer du sein MCF-7. Modifications de synthèse induites par le FGF-2.

Two-dimensional electrophoresis analysis of proteins from breast cancer cells MCF-7. Modifications of synthesis induced by FGF-2. Using high resolution two-dimensional electrophoresis, we have separated more than 1,000 proteins from the breast cancer cell line MCF-7. Computer assisted analysis of gels allowed us to classify these proteins in function of their isoelectric point, molecular weight and relative quantity. This data-base will now be used as a powerful tool to identified proteins which synthesis is regulated in various experimental or pathological situations. Thus we studied modifications induced by FGF-2. This growth factor induces the synthesis of four polypeptides which are not detected in cells not stimulated by this factor. In addition, intensity of nine other polypeptides was found increased in presence of FGF-2.

What determines the antioxidant potential of yeast cells?

Reactivity which organic radicals was compared in yeast (Saccharomyces cerevisiae) strains defective in catalase and superoxide dismutase, and with a decreased level of glutathione. Yeast cell homogenates did not show considerable strain-related differences in the ability to scavenge nitroxide (TEMPO) stable free radicals and alkoxyl free radicals generated by decomposition of the free radical initiator AAPH. The "total antioxidant status" based on scavenging of ABTS free radicals showed a good correlation with the radiation resistance of the yeasts. These results point to the importance of other factors, apart from antioxidative enzymes and glutathione, in the determination of cellular resistance to ionizing radiation and other types of free-radical stress.

Resistance to ionizing radiation and antioxidative defence in yeasts. Are antioxidant-deficient cells permanently stressed?

Radiation sensitivity of the yeast (Saccharomyces cerevisiae) strains defective in catalase and superoxide dismutase, and of decreased level of glutathione was compared. Small differences in the radiation sensitivity pointed to the highest sensitivity of the wild-type strain as compared with the antioxidant-defective mutants. Heat shock increases the radiation resistance to a different extent, the response being highest in the wild-type strain and lowest in the glutathione-deficient strain. Catalase induction by heat shock follows the same pattern, significant activity of this enzyme being detectable in the glutathione-deficient mutant under normal growth conditions. These results suggest that compensatory induction of elements of the antioxidant defence system may occur in yeast mutants deficient in antioxidants under normal growth conditions.