This study compared the in vitro responses of malignant and normal cells from the human oral cavity to tea extracts and to its main polyphenolic component, (-)-epigallocatechin gallate (EGCG). The antiproliferative effects of tea polyphenolic extracts and EGCG were more pronounced towards immortalized, tumourigenic (CAL27, HSC-2, and HSG(1)) and non-tumourigenic (S-G) cells than towards normal (GN56 and HGF-1) fibroblasts and green tea was more toxic than black tea. As the addition of tea extract or EGCG to cell culture medium led to the formation of hydrogen peroxide (H(2)O(2)), the research then focused on EGCG as an inducer of oxidative stress, using CAL27, the cancerous cells most sensitive to EGCG, HSG(1), the cancerous cells least sensitive to EGCG, and GN56 cells. The toxicity of EGCG was decreased in the presence of catalase, an enzyme that degrades H(2)O(2), or of deferoxamine, a chelator of Fe(3+). Conversely, pretreatment of the cells with the glutathione depleters, 1-chloro-2,4-dinitrobenzene and 1,3-bis(2-chloroethyl)-N-nitrosourea, potentiated the toxicity of EGCG. A 4-hr exposure to EGCG lessened the intracellular level of reduced glutathione in the CAL27 and HSG(1) cells, but not in the GN56 fibroblasts. Whereas EGCG itself did not induce lipid peroxidation, Fe(2+)-induced lipid peroxidation was potentiated by EGCG. A 72-hr exposure to cytotoxic concentrations of EGCG induced significant cytoplasmic vacuolization in all cell types. The results presented herein are consistent with EGCG acting as a prooxidant, with the cancerous cells more sensitive to oxidative stress than the normal cells.
Polyphenols of phytochemicals are thought to exhibit chemopreventive effects against cancer. These plant-derived antioxidant polyphenols have a dual nature, also acting as pro-oxidants, generating reactive oxygen species (ROS), and causing oxidative stress. When studying the overall cytotoxicity of polyphenols, research strategies need to distinguish the cytotoxic component derived from the polyphenol per se from that derived from the generated ROS. Such strategies include (a) identifying hallmarks of oxidative damage, such as depletion of intracellular glutathione and lipid peroxidation, (b) classical manipulations, such as polyphenol exposures in the absence and presence of antioxidant enzymes (i.e., catalase and superoxide dismutase) and of antioxidants (e.g., glutathione and N-acetylcysteine) and cotreatments with glutathione depleters, and (c) more recent manipulations, such as divalent cobalt and pyruvate to scavenge ROS. Attention also must be directed to the influence of iron and copper ions and to the level of polyphenols, which mediate oxidative stress.
Previous studies have demonstrated that, as naive murine CD4+ cells differentiate into Th1 cells, they lose expression of the second chain of IFN-γR (IFN-γR2). Hence, the IFN-γ-producing subset of Th cells is unresponsive to IFN-γ. Analysis of IFN-γ-producing CD8+ T cells demonstrates that, like Th1 cells, these cells do not express IFN-γR2. To define the importance of IFN-γ signaling for the development of functional CD8+ T cells, mice either lacking IFN-γR2 or overexpressing this protein were examined. While CD8+ T cell development and function appear normal in IFN-γR2−/− mice, CD8+ T cell function in IFN-γR2 transgenic is altered. IFN-γR2 transgenic CD8+ T cells are unable to lyse target cells in vitro. However, these cells produce Fas ligand, perforin, and granzyme B, the effector molecules required for killing. Interestingly, TG CD8+ T cells proliferate normally and produce cytokines, such as IFN-γ in response to antigenic stimulation. Therefore, although IFN-γ signaling is not required for the generation of normal cytotoxic T cells, constitutive IFN-γ signaling can selectively impair the cytotoxic function of CD8+ T cells.
SummaryMultidrug resistance (MDR), which is due, in part, to the overexpression of P-glycoprotein, confers resistance to a variety of natural product chemotherapeutic agents such as daunorubicin, vincristine, and colchicine. RV + cells are a P-glycoprotein overexpressing variant of the HL60 myeloid leukemia cell line. In addition to classic MDR, IkV + cells displayed relative resistance to complement-mediated cytotoxicity with both immunoglobulin G and M antibodies against different cell surface antigens, but not to antibody-dependent cellular cytotoxicity and lymphokine-activated killing. Complement resistance was reversed both by treatment with verapamil and with specific monoclonal antibodies (mAbs) capable of binding to P-glycoprotein and blocking its function. To further confirm that the resistance of IkV + cells was not a consequence of the selection of the cells on vincristine, a second system involving P-glycoprotein infectants was also investigated. K562 cells infected with the MDR1 gene, which were never selected on chemotherapeutic drugs, also displayed relative resistance to complementmediated cytotoxicity. This MDR1 infection-induced resistance was also reversed by mAbs that bind to P-glycoprotein. Therefore, the MDI< phenotype as mediated by P-glycoprotein provides resistance to complement-mediated cytotoxicity. The increased intracellular pH and the decreased membrane potential due to the MDI< phenotype may result in abnormal membrane attack complex function. This observation may have implications for the possible mechanisms of action of P-glycoprotein and for a possible physiologic role for P-glycoprotein in protection against complement-mediated autolysis.M ultidrug resistance (MDP,.) is characterized by broad resistance to several pharmacologically and chemically distinct chemotherapeutic compounds, in particular, the vinca alkaloids and the anthracyclines (1). Tumor cells that are resistant to these compounds exhibit decreased retention of drugs over time (2). The MDk phenotype (3) is the result of the overexpression of the mdrl gene product at least in part, a 170--180-kD glycoprotein known as P-glycoprotein. A "drug pump" model has been proposed in which the P-glycoprotein serves as an active transporter that pumps drugs out of the cell (1, 3, 4).The cytotoxic abilities of mAbs M195 (anti-CD33) (5) and M31 (anti-CD15) against HL60 myeloid leukemia cells and a MDt< HL60 variant that overexpresses the MDR protein (RV + [6]), were examined. In this paper, we describe the relative resistance of RV + ceils to specific mAbs that react against cell surface protein or carbohydrate targets, and mediate cytotoxicity via complement fixation. MDP,. reversal studies using verapamil and with specific mAbs to P-glycoprotein showed that a significant portion of this newly described immunological resistance is mediated by P-glycoprotein overexpression. To test whether these observations were not partly due to the selection of the tLV + cells on vincristine or a spurious phenomenon for a single cell line, K56...
showed that multidrug resistance (MDR) cells created by continuous selection with the vinca alkaloid vincristine (HL60 RV ؉ ) or by retroviral infection (K562/human MDR 1 cells) exhibited significant resistance to complement-mediated cytotoxicity (CMC). This resistance was due to the presence of overexpressed P-glycoprotein (P-GP). In this paper, we probe the molecular mechanism of this phenomenon. We test whether the significant elevated intracellular pH (pH i ) that accompanies P-GP overexpression is sufficient to confer resistance to CMC and whether this resistance is related to effects on complement function in the cell membrane. Control HL60 cells not expressing P-GP, but comparably elevated in cytosolic pH i by two independent methods (CO 2 "conditioning" or isotonic Cl؊ substitution), are tested for CMC using two different antibody-antigen systems (human IgG and murine IgM; protein and carbohydrate) and two complement sources (rabbit and human). Elevation of pH i by either of these methods or by expression of P-GP confers resistance to CMC. Resistance is not observed when the alkalinization mediated by reverse Cl
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