Interfering with ROS metabolism in cancer cells: the potential role of quercetin.

Abstract: A main feature of cancer cells, when compared to normal ones, is a persistent pro-oxidative state that leads to an intrinsic oxidative stress. Cancer cells have higher levels of reactive oxygen species (ROS) than normal cells, and ROS are, in turn, responsible for the maintenance of the cancer phenotype. Persistent ROS stress may induce adaptive stress responses, enabling cancer cells to survive with high levels of ROS and maintain cellular viability. However, excessive ROS levels render cancer cells highly susceptible to quercetin, one of the main dietary flavonoids. Quercetin depletes intracellular glutathione and increases intracellular ROS to a level that can cause cell death

Quercetin and Cancer Chemoprevention

Several molecules present in the diet, including flavonoids, can inhibit the growth of cancer cells with an ability to act as “chemopreventers”. Their cancer-preventive effects have been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis as well as the antioxidant functions. The antioxidant activity of chemopreventers has recently received a great interest, essentially because oxidative stress participates in the initiation and progression of different pathological conditions, including cancer. Since antioxidants are capable of preventing oxidative damage, the wide use of natural food-derived antioxidants is receiving greater attention as potential anti-carcinogens. Among flavonoids, quercetin (Qu) is considered an excellent free-radical scavenging antioxidant, even if such an activity strongly depends on the intracellular availability of reduced glutathione. Apart from antioxidant activity, Qu also exerts a direct, pro-apoptotic effect in tumor cells, and can indeed block the growth of several human cancer cell lines at different phases of the cell cycle. Both these effects have been documented in a wide variety of cellular models as well as in animal models. The high toxicity exerted by Qu on cancer cells perfectly matches with the almost total absence of any damages for normal, non-transformed cells. In this review we discuss the molecular mechanisms that are based on the biological effects of Qu, and their relevance for human health

Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry.

Reactive oxygen species (ROS) are continuously produced in the cell as a consequence of aerobic metabolism, and are controlled by several antioxidant mechanisms. An accurate measurement of ROS is essential to evaluate the redox status of the cell, or the effects of molecules with the pro-oxidant or antioxidant activity. Here we report a cytofluorimetric technique for measuring simultaneously, at the single-cell level, hydrogen peroxide and superoxide anion, reduced glutathione (a main intracellular antioxidant) and cell viability. The staining is performed with the fluorescent dyes 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA), hydroethidine (HE), monobromobimane (MBB) and TO-PRO-3. This analysis is possible with new-generation flow cytometers equipped with several light sources (in our case, four lasers and an UV lamp), which excite different fluorochromes. This approach is extremely useful to study the balance between ROS content and antioxidants in cells receiving different stimuli, and to analyze the relationship between oxidative stress and cell death

Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry.

Reactive oxygen species (ROS) are continuously produced in the cell as a consequence of aerobic metabolism, and are controlled by several antioxidant mechanisms. An accurate measurement of ROS is essential to evaluate the redox status of the cell, or the effects of molecules with the pro-oxidant or antioxidant activity. Here we report a cytofluorimetric technique for measuring simultaneously, at the single-cell level, hydrogen peroxide and superoxide anion, reduced glutathione (a main intracellular antioxidant) and cell viability. The staining is performed with the fluorescent dyes 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA), hydroethidine (HE), monobromobimane (MBB) and TO-PRO-3. This analysis is possible with new-generation flow cytometers equipped with several light sources (in our case, four lasers and an UV lamp), which excite different fluorochromes. This approach is extremely useful to study the balance between ROS content and antioxidants in cells receiving different stimuli, and to analyze the relationship between oxidative stress and cell death

T cell homeostasis in centenarians: from the thymus to the periphery.

The immune system undergoes a process of profound remodelling during aging, referred to as immunosenescence, and characterized by complex modifications of several components. In this review, we discuss recent developments and observations regarding the generation of T cells in the thymus during aging and longevity, and the regulation and maintenance of peripheral blood lymphocytes. The generation of new T cells is indeed crucial to maintain a functional immune system, and is a fundamental step to avoid unsuccessful aging, thus reaching longevity in good health. Mechanisms will be described that are related to the production and maintenance of those lymphocytes defined "recent thymic emigrants", and to the detection of the so called "T cell receptor rearrangement excision circles (TREC)", along with the presence in the periphery of na\uefve and memory T cells, that can be influenced and regulated by several different mechanisms. Several strategies aimed at improving thymic functionality are currently receiving a growing interest, and some of them are based on molecules that are produced by, and/or act on immune cells. Data on the possible use of these molecules, including cytokines like interleukin (IL)-7, IL-15 and keratinocyte growth factor, to restore thymic function are reviewed and discussed

Quercetin and cancer chemoprevention.

Several molecules present in the diet, including flavonoids, can inhibit the growth of cancer cells with an ability to act as "chemopreventers". Their cancer-preventive effects have been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis as well as the antioxidant functions. The antioxidant activity of chemopreventers has recently received a great interest, essentially because oxidative stress participates in the initiation and progression of different pathological conditions, including cancer. Since antioxidants are capable of preventing oxidative damage, the wide use of natural food-derived antioxidants is receiving greater attention as potential anti-carcinogens. Among flavonoids, quercetin (Qu) is considered an excellent free-radical scavenging antioxidant, even if such an activity strongly depends on the intracellular availability of reduced glutathione. Apart from antioxidant activity, Qu also exerts a direct, pro-apoptotic effect in tumor cells, and can indeed block the growth of several human cancer cell lines at different phases of the cell cycle. Both these effects have been documented in a wide variety of cellular models as well as in animal models. The high toxicity exerted by Qu on cancer cells perfectly matches with the almost total absence of any damages for normal, non-transformed cells. In this review we discuss the molecular mechanisms that are based on the biological effects of Qu, and their relevance for human health

NK-cell phenotype at interruption underlies widely divergent duration of CD4+-guided antiretroviral treatment interruption.

Long-term side effects may represent a relevant burden of antiretroviral treatment (ART) in HIV-infected patients with good CD4 immune reconstitution over extended time spans. CD4-guided treatment interruption (TI) has been evaluated to address this point and may result in a wide spectrum of time off ART in different patient cohorts. We studied whether differences in innate immune responses, in particular NK cells, are associated to patterns of longer (LoTI) or a shorter (ShTI) TI. Clinical cohort parameters were analyzed on a group of patients widely diverging for TI duration (18 months) on samples before TI, including NK-cell analysis and function by natural cytotoxicity receptor (NCR)-triggered γ-IFN production. Although persistently reduced NCR expression (NKp30) and function were observed in both LoTI and ShTI patients on ART compared with healthy donors, relevant differences were observed at baseline TI in those patients who subsequently developed LoTI course. Lower expression of NKG2D and NKp46 on NK cells. This also translates in reduced γ-IFN production in redirected functional assays. Thus, differences in innate immune balance exist during ART, may be associated to differential control of HIV infection and their understanding could explain clinical differences in individual patients that are not reflected by CD4(+) cell counts alone

Cytotoxic granule release dominates gag-specific CD4+ T-cell response in different phases of HIV infection.

BACKGROUND: The activity of virus-specific T lymphocytes, among which those capable of a polyfunctional response against the viral protein gag, is crucial to control HIV infection. OBJECTIVE: The objective of this study is to investigate the polyfunctionality of gag-specific T cells in different phases of HIV infection, analyzing markers related to T-helper cell 1 (Th1) and degranulation/cytotoxicity, and the production of Th1 cytokines in peripheral blood lymphocytes from patients experiencing an acute primary infection, long-term nonprogressors, patients naive for antiretroviral drugs, and patients taking HAART. MATERIALS AND METHODS: Cells were stimulated with a pool of gag-derived peptides or with a superantigen (staphylococcal enterotoxin B). Using eight-color polychromatic flow cytometry, we analyzed the expression of interleukin-2, interferon-gamma, CD154, and CD107a by CD4 and CD8 T cells. RESULTS: The main finding was that in all HIV-positive patients, about half gag-specific CD4 T cells were CD107a, that is, able to degranulate. CD4CD154 cells unable to produce Th1 cytokines were the second most represented population. Truly polyfunctional CD4 T cells were very rare and present only in a few long-term nonprogressors. Superantigen stimulation showed that CD4 T lymphocytes from all patients displayed a typical Th response, including interleukin-2 and interferon-gamma production, lacking CD107a expression. CONCLUSION: In all the aforementioned phases of HIV infection, the large majority of gag-specific CD4 T lymphocytes cannot be identified by the sole expression of interleukin-2 and interferon-gamma, which is early impaired. Degranulation and helper functions other than Th1 cytokine production are the predominant features of HIV-specific CD4 lymphocytes

CD4+ T-cell differentiation, regulatory T cells and gag-specific T lymphocytes are unaffected by CD4-guided treatment interruption and therapy resumption.

OBJECTIVES:Despite limiting exposure to antiretroviral drugs, structured treatment interruptions can influence multiple aspects of T-cell immunity, particularly those regarding CD4(+) T lymphocytes. We evaluated the impact of CD4-guided treatment interruption (CD4-GTI) and treatment resumption on regulatory T cells (Tregs), T-lymphocyte activation, differentiation and polyfunctional gag-specific response.METHODS:Patients were analyzed just prior to treatment interruption, at 2 and 6 months after treatment interruption, just prior to treatment resumption and at 2 and 6 months after treatment resumption. Thawed peripheral blood mononuclear cells were stained immediately for phenotype analysis or stimulated with HIV-gag peptides and analyzed by polychromatic flow cytometry.RESULTS:Treatment interruption resulted in a CD4(+) cell count decrease and plasma viral load (pVL) increase, but did not preclude a good immune reconstitution and a complete suppression of pVL after treatment resumption. Treatment interruption did not influence CD4(+) T-cell differentiation and Treg subsets. During treatment interruption, gag-specific CD4(+) T cells were not lost, although the frequency of HIV-specific CD8(+) cells increased. Most gag-specific CD4(+) T cells were potentially cytotoxic (CD107a(+)) and were not influenced by pVL or by HAART. Most helper (CD154(+)) gag-specific CD4(+) T lymphocytes did not produce interferon-γ or interleukin-2.CONCLUSION:CD4-GTI did not cause depletion of memory cells, Tregs or HIV-specific CD4(+) cells and, on the contrary, could induce HIV-specific responses. If guided by CD4(+) T-cell count, treatment interruption does not provoke irreversible immune damages