Differential Cytotoxic Activity Research Articles

Differential cytotoxic and prooxidnant activity of knipholone and knipholone anthrone

Knipholone (KP) and knipholone anthrone (KA) are natural 4-phenylanthraquinone structural analogues with established differential biological effects including in vitro antioxidant [1] and antimicrobial properties [2]. The present study was designed to investigate the comparative in vitro cytotoxic activity and the possible mechanism of action of these two compounds. We demonstrated that KA is by order of magnitude more cytotoxic to mammalian cells than KP. In parallel with the demonstrated cytotoxic effect, KA but not KP induces prooxidative DNA damage in the presence of copper ions. In order to establish the possible involvement of reactive oxygen species in the KA-mediated prooxidative effect, we investigated the protective effect of several metal chelators and reactive oxygen species scavengers. Our data suggest that reactive oxygen species such as hydrogen peroxide are involved and a good correlation between prooxidative action, antioxidant effect and cytotoxicity is established for these two structural analogues. The chemistry, pharmacology and potential medicinal/toxicological potential of these compounds are discussed.

Functional role of human NK cell receptor 2B4 (CD244) isoforms

2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM/CD150), is expressed on all NK cells, a subpopulation of T cells, monocytes and basophils. Human NK cells express two isoforms of 2B4, h2B4-A and h2B4-B that differ in a small portion of the extracellular domain. In the present investigation, we have studied the functions of h2B4-A and h2B4-B. Our study demonstrated that these two isoforms differ in their binding affinity for CD48, which results in differential cytotoxic activity as well as intracellular calcium release by NK cells upon target cell recognition. Analysis of the predicted 3-D structure of the two isoforms showed conformational differences that could account for their differences in binding affinity to CD48. h2B4-A was able to mediate natural cytotoxicity against CD48-expressing K562 target cells and induce intracellular calcium release, whereas h2B4-B showed no effects. NK-92MI, U937, THP-1, KU812, primary monocytes, basophils and NK cells showed expression of both h2B4-A and h2B4-B whereas YT and IL-2-activated NK cells did not show any h2B4-B expression. Stimulation of NK cells through 2B4 resulted in decreased mRNA levels of both h2B4-A and h2B4-B indicating that down-regulation of 2B4 isoforms may be an important factor in controlling NK cell activation during immune responses.

Functional role of human natural killer cell receptor 2B4 (CD244) isoforms

Natural killer (NK) cells are a subpopulation of lymphocytes that represent the first line of defense against stressed, virally infected and malignant cells. NK cell functions are controlled by a balance between positive and negative signals through various receptors. We have identified, cloned and characterized the 2B4 (CD244) receptor in mice and human. 2B4 interaction with its ligand CD48 regulates NK, T and B cell functions and thus plays an important role in various immune responses. Human NK cells express two isoforms of 2B4, h2B4‐A and h2B4‐B that differ in a small portion of the extracellular domain. In the present investigation, we have studied the functions of h2B4‐A and h2B4‐B. Our data demonstrate that these two isoforms differ in their binding affinity for CD48 and this result in differential cytotoxic activity by NK cells against tumor targets. h2B4‐A was able to mediate natural cytotoxicity against CD48 expressing K562 target cells confirming that h2B4‐A is an activating receptor, whereas h2B4‐B showed no effects. Analysis of the predicted 3‐D structure of the two isoforms showed conformational differences which may be responsible for their differences in binding affinity to CD48. Thus differential expression of 2B4 isoforms by NK cells may regulate immune response mediated through 2B4‐CD48 interactions. This work was supported by the NIH grant CA85753.

Effects of the antitumoural dequalinium on NB4 and K562 human leukemia cell lines: Mitochondrial implication in cell death

Dequalinium (DQA) is a delocalized lipophylic cation that selectively targets the mitochondria of carcinoma cells. However, the underlying mechanisms of DQA action are not yet well understood. We have studied the effects of DQA on two different leukemia cell lines: NB4, derived from acute promyelocytic leukemia, and K562, derived from chronic myeloid leukemia. We found that DQA displays differential cytotoxic activity in these cell lines. In NB4 cells, a low DQA concentration (2 μM) induces a mixture of apoptosis and necrosis, whereas a high DQA concentration (20 μM) induces mainly necrosis. However, K562 cell death was always by necrosis as the cells showed a resistance to apoptosis at all time-periods and DQA concentrations assayed. In both cell lines, the cell death seems to be mediated by alterations of mitochondrial function as evidenced by loss of mitochondrial transmembrane potential, O 2 − accumulation and ATP depletion. The current study improves the knowledge on DQA as a novel anticancer agent with a potential application in human acute promyelocytic leukemia chemotherapy.

Cytotoxic Activities of Snow Lotus Filtrates Against Human Cancer Cell Lines and Origin of Active Factor(s)

Snow Lotus (SL) filtrate was prepared by incubation of SL grains with pasteurized and unboiled 4% milk for 24 h at room temperature and tested on cultured human cancer cells, namely, human colon adenocarcinoma (HCA), HepG2, MCF-7 and compared with a normal mouse (NCTC clone 929) cell line. It was observed that this filtrate exhibited cytotoxic activities in HCA (LD50 16.6 µl/ml) and HepG2 (LD50 17.1 µl/ml) cells and to a lesser extent in MCF-7 (LD50 28.1 µl/ml) and normal mouse cells (LD50 24.3 µl/ml). Different SL filtrates were also prepared by incubation of grains with 2, 1 and 0% fat content milks. These filtrates exhibited moderate to high cytotoxic activities. Interestingly, the filtrate obtained from 2% milk showed significant differential cytotoxic activity in the range of 39 to 44% between human cancer and mouse cell lines. The cytotoxic studies were also carried out with filtrate of shorter incubation period, where no cytotoxic activity observed. Similarly, SL water filtrate was also inactive in all cell cultures. The origin of cytotoxic factor(s) in the filtrates is discussed.

In vitro activity of dimethylarsinic acid against human leukemia and multiple myeloma cell lines.

Arsenic trioxide (As(2)O(3)), an inorganic arsenic compound, has recently been approved for the treatment of relapsed or refractory acute promyelocytic leukemia. However, systemic toxicity associated with As(2)O(3) treatment remains a problem. Inorganic arsenic is detoxified in vivo by methylation reactions into organic arsenic compounds that are less toxic. We investigated the antiproliferative and cytotoxic activity of dimethylarsinic acid (DMAA), an organic arsenic derivative and major metabolic by-product of As(2)O(3), against a panel of eight leukemia and multiple myeloma cell lines. As(2)O(3) was tested in comparison. In clonogenic assay, the average concentration of DMAA that suppressed cell colony growth by 50% was 0.5-1 m M, while for As(2)O(3) it was on average 1-2 microM. At those concentrations DMAA and As(2)O(3) had significantly less effect on colony growth of normal progenitor cells. Cytotoxic doses of DMAA and As(2)O(3) in 3-day trypan blue dye exclusion assay experiments were similar to doses effective in clonogenic assay. Assessment of apoptosis by annexin V assay revealed a high rate of apoptosis in all cell lines treated with DMAA and As(2)O(3), but significantly less effect on normal progenitor cells. DMAA, unlike As(2)O(3), had no effect on the maturation of leukemic cells. DMAA exerts differential antiproliferative and cytotoxic activity against leukemia and multiple myeloma cells, with no significant effect on normal progenitor cells. However, concentrations of DMAA needed to achieve such efficacy are up to 1000 times those of As(2)O(3). Evaluation of novel organic arsenic that would combine the high efficacy of As(2)O(3) and the low toxicity of DMAA is warranted.

Selective toxicity of the tricyclic thiophene NSC 652287 in renal carcinoma cell lines: Differential accumulation and metabolism

The tricyclic compound 2,5-bis(5-hydroxymethyl-2-thienyl)furan (NSC 652287) has shown a highly selective pattern of differential cytotoxic activity in the tumor cell lines comprising the National Cancer Institute (NCI) Anticancer Drug Screen. The mechanism underlying the selective cytotoxicity is unknown. We hypothesized that differential sensitivity to the compound observed in several renal tumor cell lines could be the result of selective accumulation or differential metabolism of this agent. We demonstrated here that the capacity of certain renal cell lines to accumulate and retain the compound, determined by accumulation of [ 14C]NSC 652287-derived radioactivity and by flow cytometric determination of unlabeled compound, paralleled the sensitivity of the renal cell lines to growth inhibition by NSC 652287: A-498 > TK-10 ≫ ACHN ∼ UO-31. The ability of the cell lines to metabolize [ 14C]NSC 652287 to a reactive species capable of binding covalently to cellular macromolecules also directly correlated with sensitivity to the compound. Different patterns of metabolites were generated by relatively more drug-sensitive cell lines in comparison with drug-resistant cell lines. The metabolizing capacity for NSC 652287 was localized primarily to the cytosolic (S100) fraction. The rate of metabolism in the cytosolic fraction from the most sensitive renal cell line, A-498, was faster than that observed in the cytosolic fractions from the other, less sensitive cell lines. The data support the hypothesis that both selective cellular accumulation and the capacity to metabolize NSC 652287 to a reactive species by certain renal carcinoma cell types are the basis for the differential cytotoxicity of this compound class.

11-oxygenated cytotoxic 8,9-secokauranes from a New Zealand liverwort, Lepidolaena taylorii.

Four new cytotoxic 8,9-secokauranes have been identified from the liverwort Lepidolaena taylorii. The 11-oxygenation found in three of these has not been encountered in the 8,9-secokauranes known from higher plants. NMR studies were combined with molecular modelling to determine the preferred conformations. Six structurally related kauren-15-ones were also found, including three new compounds. Some of these compounds showed differential cytotoxic activity against human tumor cell lines. The probable mode of cytotoxic action was supported by Michael addition of a thiol. Two 8,9-secokauranes were the main cytotoxins in another New Zealand liverwort. L. palpebrifolia.

Cytotoxic 8,9-secokaurane diterpenes from a New Zealand liverwort, Lepidolaena taylorii

The main cytotoxic compound from the liverwort Lepidolaena taylorii is the known ent-8,9-seco-7-hydroxy-kaura-8(14),16-dien-9,15-dione 1. The relative stereochemistry of a new cytotoxic compound, 16,17-dihydro derivative 2, was supported by molecular modelling. Another new cytotoxic compound was identified as 8,14-epoxide 3 by preparation from 1. These compounds showed differential cytotoxic activity against human tumor cell lines.

Intrinsic Cytotoxicity of Natural Killer Cells to Pancreatic Islets In Vitro

BB rats develop spontaneous autoimmune insulin-dependent diabetes mellitus that is similar to human insulin-dependent diabetes. In this study, we used an in vitro islet cell cytotoxicity assay to study the possible role of natural killer (NK) cells and their soluble effector molecules in this disorder. First, the results demonstrated that in vivo treatment of acutely diabetic BB rats with anti-asialogangliosideM1 (an NK cell antiserum) but not with anti-T-lymphocyte antibodies reduces spleen cell cytotoxic activity to islets in vitro. Flow microfluorometry (FMF)-sorting experiments were then used to confirm that the splenic cytotoxic effector cell in acutely diabetic BB rats is a CD8+/CD5- NK cell. Further analysis demonstrated that both FMF-sorted NK cell populations from Wistar-Furth rats and unfractionated spleen cells from athymic nu/nu rats with high intrinsic NK cell activity also exhibit high islet cell cytotoxic activity in vitro. Finally, we found that the kinetics and differential cytotoxic activity of NK cells toward islets in vitro could be mimicked by NK cell culture supernatants containing high levels of NK cytotoxic factor (NKCF). The islet cytotoxic activity of these culture supernatants was specifically inhibited by the addition of anti-NKCF monoclonal antibody. These results demonstrate that NK cells from diabetic and nondiabetic rats are cytotoxic to islet cells in vitro. They further suggest that this cytotoxic effect may be mediated in part through the production and release of soluble factors such as NKCF.

Efficacy of Ex Vivo Purging for Autologous Bone Marrow Transplantation in the Treatment of Acute Nonlymphoblastic Leukemia

AbstractWe used an in vitro measure of drug activity to predict the efficacy of ex vivo purging of leukemic cells from autologous bone marrow grafts. We previously found that the myeloid progenitor cell (CFU-GM) content of the marrow grafts after ex vivo purging with 4-hydroperoxycyclophos- phamide (4-HC) correlates with time to hematologic recovery after autologous bone marrow transplantation in patients with acute nonlymphoblastic leukemia. We observed that variable red blood cell concentration of the bone marrow incubation mixture results in differential cytotoxic activity of 4-HC. The CFU-GM content of the graft after the ex vivo treatment is a measure of this 4-HC activity. We analyzed the disease-free survival of 45 patients with acute nonlymphoblastic leukemia undergoing autologous bone marrow transplantation with 4-HC purged grafts. Patients who relapsed after transplantation had 4.2 ± 1.1% of graft CFU-GM surviving the ex vivo purge, compared with 1.1 ± 0.4% for patients who achieved a sustained remission (P = .06). Twenty-three patients with a CFU-GM content after 4-HC purging of >1% of the pretreatment value had an actuarial disease-free survival of 12%, compared to 36% for 22 patients with a ≤1% CFU-GM content after purging (P = .006). Therefore, percent CFU-GM survival as a measure of 4-HC cytotoxicity identified a group of patients with insufficient purging. Although no randomized clinical trials have documented the need for ex vivo purging, our results suggest that effective bone marrow purging is important for the optimal application of autologous transplantation in the treatment of acute nonlymphoblastic leukemia.

Efficacy of ex vivo purging for autologous bone marrow transplantation in the treatment of acute nonlymphoblastic leukemia

We used an in vitro measure of drug activity to predict the efficacy of ex vivo purging of leukemic cells from autologous bone marrow grafts. We previously found that the myeloid progenitor cell (CFU-GM) content of the marrow grafts after ex vivo purging with 4- hydroperoxycyclophosphamide (4-HC) correlates with time to hematologic recovery after autologous bone marrow transplantation in patients with acute nonlymphoblastic leukemia. We observed that variable red blood cell concentration of the bone marrow incubation mixture results in differential cytotoxic activity of 4-HC. The CFU-GM content of the graft after the ex vivo treatment is a measure of this 4-HC activity. We analyzed the disease-free survival of 45 patients with acute nonlymphoblastic leukemia undergoing autologous bone marrow transplantation with 4-HC purged grafts. Patients who relapsed after transplantation had 4.2 +/- 1.1% of graft CFU-GM surviving the ex vivo purge, compared with 1.1 +/- 0.4% for patients who achieved a sustained remission (P = .06). Twenty-three patients with a CFU-GM content after 4-HC purging of greater than 1% of the pretreatment value had an actuarial disease-free survival of 12%, compared to 36% for 22 patients with a less than or equal to 1% CFU-GM content after purging (P = .006). Therefore, percent CFU-GM survival as a measure of 4-HC cytotoxicity identified a group of patients with insufficient purging. Although no randomized clinical trials have documented the need for ex vivo purging, our results suggest that effective bone marrow purging is important for the optimal application of autologous transplantation in the treatment of acute nonlymphoblastic leukemia.

Differential cytotoxic activity of potassium dichromate on nucleoside uptake in BHK fibroblasts

In cultures of hamster fibroblasts (BHK cell line) treated with potassium dichromate (K 2Cr 2O 7) nucleic acid and protein syntheses are differentially inhibited, and nucleoside uptake into the intracellular pool is characterized by a stimulation phase followed by an inhibition phase. Different patterns are observed for the uptake of each ribo- and deoxyribonucleoside, pyrimidine nucleoside (particularly deoxycytidine) uptake reaching the highest stimulation level. Kinetics of thymidine and deoxycytidine initial uptake at different exogenous nucleoside concentrations show that Ka 2Cr 2O 2 affects both simple and facilitated diffusion of nucleosides. The time course of thymidine and deoxycytidine pool saturation suggests however that the effects of K 2Cr 2O 7 on plasma membrane permeability are partially counterbalanced by modifications of pool size deriving from the concomitant alteration of steps of nucleoside metabolism separate from nucleoside uptake.

Differential cytotoxic activity of anticollagen serum on rat osteoblasts and fibroblasts in tissue culture

Rat skin fibroblasts grown in tissue culture were lysed by anti-rat-tail collagen serum and antibodies to the ordered collagen-like synthetic polymer (Pro-Gly-Pro) n. This cytotoxic effect is complement-dependent and occurs only if the fibroblasts were pretreated with trypsin. These anti-sera have very little cytotoxicity on cultured rat osteoblasts. This differential cytotoxicity is not due to differential binding of anticollagen serum to the cells. Both osteoblasts and skin fibroblasts bind the anticollagen serum as was demonstrated by fluorescent immunoglobulins.