Curcuminoid Binding to Embryonal Carcinoma Cells: Reductive Metabolism, Induction of Apoptosis, Senescence, and Inhibition of Cell Proliferation

Curcumin preparations typically contain a mixture of polyphenols, collectively referred to as curcuminoids. In addition to the primary component curcumin, they also contain smaller amounts of the co-extracted derivatives demethoxycurcumin and bisdemethoxycurcumin. Curcuminoids can be differentially solubilized in serum, which allows for the systematic analysis of concentration-dependent cellular binding, biological effects, and metabolism. Technical grade curcumin was solubilized in fetal calf serum by two alternative methods yielding saturated preparations containing either predominantly curcumin (60%) or bisdemethoxycurcumin (55%). Continual exposure of NT2/D1 cells for 4–6 days to either preparation in cell culture media reduced cell division (1–5 µM), induced senescence (6–7 µM) or comprehensive cell death (8–10 µM) in a concentration-dependent manner. Some of these effects could also be elicited in cells transiently exposed to higher concentrations of curcuminoids (47 µM) for 0.5–4 h. Curcuminoids induced apoptosis by generalized activation of caspases but without nucleosomal fragmentation. The equilibrium binding of serum-solubilized curcuminoids to NT2/D1 cells incubated with increasing amounts of curcuminoid-saturated serum occurred with apparent overall dissociation constants in the 6–10 µM range. However, the presence of excess free serum decreased cellular binding in a hyperbolic manner. Cellular binding was overwhelmingly associated with membrane fractions and bound curcuminoids were metabolized in NT2/D1 cells via a previously unidentified reduction pathway. Both the binding affinities for curcuminoids and their reductive metabolic pathways varied in other cell lines. These results suggest that curcuminoids interact with cellular binding sites, thereby activating signal transduction pathways that initiate a variety of biological responses. The dose-dependent effects of these responses further imply that distinct cellular pathways are sequentially activated and that this activation is dependent on the affinity of curcuminoids for the respective binding sites. Defined serum-solubilized curcuminoids used in cell culture media are thus suitable for further investigating the differential activation of signal transduction pathways

Inhibition of human lymphocyte-mediated mitogen-induced cytotoxicity of murine L-929 cells by heterologous anti-human lymphotoxin antisera in vitro.

Heterologous anti-human lymphotoxin (LT) antisera have been employed to investigate the role of LT in mitogen-(Con-A, PHA) induced destruction of murine L-929 cells by human lymphocytes in vitro. These various antisera will effectively neutralize human LT molecules associated with the stable (70 to 90,000 dalton) alpha-LT class of cytotoxin (anti-alpha-LT), the more unstable (35 to 50,000 dalton) beta-LT class of cytotoxins (anti-beta-LT), and antisera which will neutralize all classes of these cytotoxins in vitro, anti-whole supernatant (anti-W.S.). These anti-LT sera will greatly inhibit lysis of L-929 cells by using mitogen-activated human effector lymphocytes in vitro. This blocking was shown to be mediated by whole serum, purified IgG, or IgG-Fab fragments, which had been extensively absorbed with bovine serum, human serum, mitogens, and normal human lymphocytes. Inhibition of lysis was not apparently due to interference with either lymphocyte-target cell contact or lymphocyte activation step(s). The blocking effects of these sera were also shown to occur during the lymphocyte-independent phase of the lytic reaction. These data support the concept that the lymphocyte deposits an LT-like effector molecule on the target-L cell surface during the lymphocyte-dependent phase, which mediates cell lysis at a later time during the lymphocyte-independent phase

In vitro lymphocyte cytotoxicity. II. Unstable lymphotoxins (beta-LT) secreted and inactivated by mitogen-stimulated human lymphocytes.

Supernatants from phytohemagglutinin (PHA)-activated human lymphocytes contain two major classes of cytotoxins (α-LT and β-LT). While α-LT appears to be stable, and the major component in 5-day culture supernatants, the majority of cytolytic activity at earlier intervals in these cultures is due to a "family" of highly unstable cytotoxins which are both secreted and destroyed at a rapid rate. The inactivation of the unstable LT molecules appears to be due to: (a) inherent instability o β-LT molecules, and (b) a lymphocyte-mediated inactivation mechanism(s) which involves serum. © 1976

Inhibition of human lymphocyte-mediated mitogen-induced cytotoxicity of murine L-929 cells by heterologous anti-human lymphotoxin antisera in vitro.

Heterologous anti-human lymphotoxin (LT) antisera have been employed to investigate the role of LT in mitogen-(Con-A, PHA) induced destruction of murine L-929 cells by human lymphocytes in vitro. These various antisera will effectively neutralize human LT molecules associated with the stable (70 to 90,000 dalton) alpha-LT class of cytotoxin (anti-alpha-LT), the more unstable (35 to 50,000 dalton) beta-LT class of cytotoxins (anti-beta-LT), and antisera which will neutralize all classes of these cytotoxins in vitro, anti-whole supernatant (anti-W.S.). These anti-LT sera will greatly inhibit lysis of L-929 cells by using mitogen-activated human effector lymphocytes in vitro. This blocking was shown to be mediated by whole serum, purified IgG, or IgG-Fab fragments, which had been extensively absorbed with bovine serum, human serum, mitogens, and normal human lymphocytes. Inhibition of lysis was not apparently due to interference with either lymphocyte-target cell contact or lymphocyte activation step(s). The blocking effects of these sera were also shown to occur during the lymphocyte-independent phase of the lytic reaction. These data support the concept that the lymphocyte deposits an LT-like effector molecule on the target-L cell surface during the lymphocyte-dependent phase, which mediates cell lysis at a later time during the lymphocyte-independent phase

In vitro lymphocyte cytotoxicity. II. Unstable lymphotoxins (beta-LT) secreted and inactivated by mitogen-stimulated human lymphocytes.

Supernatants from phytohemagglutinin (PHA)-activated human lymphocytes contain two major classes of cytotoxins (α-LT and β-LT). While α-LT appears to be stable, and the major component in 5-day culture supernatants, the majority of cytolytic activity at earlier intervals in these cultures is due to a "family" of highly unstable cytotoxins which are both secreted and destroyed at a rapid rate. The inactivation of the unstable LT molecules appears to be due to: (a) inherent instability o β-LT molecules, and (b) a lymphocyte-mediated inactivation mechanism(s) which involves serum. © 1976

The LT system in experimental animals. II. Physical and immunologic characteristics of molecules with LT activity rapidly released by murine lymphoid cells activated on lectin-coated allogeneic monolayers in vitro.

The present studies investigate the physicochemical and immunologic properties of cell-lytic molecules released in vitro by nonadherent C57/BL/6 splenocytes or nylon wool-enriched T cell populations activated on monolayers of PHA coated L-929 cells. The findings reveal that cell-lytic molecules released by these lymphoid cells are physically heterogeneous. These molecules can be separated by gel filtration into similar m.w. classes previously observed for human lymphotoxin (LT) molecules. Three major classes, termed complex (Cx) (>200,000 d), α heavy [α(H)] (110 to 140,000 d), α light [α(L)] (60 to 90,000 d), and two minor classes, β at 40 to 50,000 d and γ at 10 to 20,000 d, were observed. Chromatography of supernatants in high ionic strength buffers dissociated Cx and α(H) to the smaller m.w. α(L) form. This evidence suggests that Cx and α(H) MW classes are physically related to the smaller m.w. α(L) class. Fractionation of the α(H) m.w. LT class by DEAE or PAGE resolved these molecules into additional distinct subclasses. Antisera were made against fresh serum-free whole supernatants (anti-WS) or rechromatographed Ultrogel fractions containing α(H) molecules [anti-α(H)]. Anti-α(H) and anti-WS react with all m.w. classes of murine LT molecules, indicating these various forms are immunologically related. These antisera do not react with LT molecules obtained from several other animal species or with 'nonspecific' intracellular toxins, e.g., lysosomal enzymes, present in normal PMN or phagocytic cells. These data indicate that materials with cell-lytic activity present in these culture supernatants are LT molecules, because: a) certain m.w. forms observed are similar to those reported previously, and b) these various m.w. forms are all physically and immunologically interrelated. These studies also indicate that murine LT molecules like human LT molecules are heterogeneous, but appear to comprise a system of subunits, in which the large m.w. form may dissociate into the smaller m.w. forms

The LT system in experimental animals. I. Rapid release of high levels of lymphotoxin (LT) activity from murine lymphocytes during the interaction with lectin-treated allogeneic or xenogeneic target cells in vitro.

High levels of cytotoxic activity (lymphotoxin-like (LT) detectable on L-929 cells was obtained in serum-free culture supernatants when non-adherent murine splenic lymphocytes or nylon wool-enriched T cells were cultured on monolayers of mitogen- (PHA) coated allogeneic or xenogeneic stimulator cells in vitro. Levels of lytic activity were lower in supernatants obtained from splenocyte cultures containing glass-adherent cell populations. Although release of lytic activity was very rapid, reaching maximal levels by 6 to 10 hr, this activity was very unstable. The levels of toxic activity in 8-hr supernatants were 20 to 50 times the levels obtained when lymphocytes were cultured with various dosages of mitogen (PHA-P, Con A) alone, even after 5 days of incubation. This phenomenon was not unique to murine lymphoid cells, for similarly high levels of LT activity were found in supernatants from lymphoid cells obtained from several animal species activated in a similar fashion. These results indicate that lymphoid cells from several animal species are capable of rapidly releasing high levels of cell-lytic activity in vitro not previously noted, and provide a means for obtaining highly active supernatants for biochemical studies. Furthermore, the data suggest that rapid release of LT may depend upon the nature of the cellular activating stimulus involving interaction of the lymphocyte with both lectin and target cell surface

In vitro lymphocyte cytotoxicity. I. Evidence of multiple cytotoxic molecules secreted by mitogen activated human lymphoid cells in vitro.

Multiple families of cytotoxic molecules [Lymphotoxin (LT)] have been identified in phytohemagglutinin (PHA-P) activated human lymphocyte supernatants and lymphocyte homogenates, using gel filtration chromatography on Sephadex G-150. These macromolecules have molecular weights of 80-90,000, 50,000, and 10-15,000 daltons and have been termed LT , LT and LT , respectively. They are secreted by cells from a variety of lympboid tissues, i.e., tonsil, adenoid, and peripheral blood. The kinetics of appearance of the cytotoxins indicate that all three are present within 16 hr after lymphocyte activation. However, while LT and LT persist in these cultures through day 5, LT is not detectable after day 3. These molecules can also be detected when either PHA or concanavalin A are employed as the stimulating agent. Moreover, the relative amounts of LT , LT and LT activity in a given supernatant vary dramatically from culture to culture. Extracellular levels of LT accumulate and peak by 4 to 5 days in culture, however, intracellular levels of LT reach a maximum on day 3 and decrease to very low levels on day 5. Mitogen-stimulated lymphocytes at 3 days contain intracellular levels of LT which are several logs higher than that detectable in unstimulated cells. This observation suggests that both the biosynthesis and secretion of lymphotoxin is governed by a regulatory control process(es). © 1976. 2 2 3 1 2 3 1 2