Doxorubicin affects tau protein metabolism in human neuroblastoma cells

The microtubule associated protein called tau, found primarily in neurons, was detected in a human neuroblastoma cell line, LAN-5. Cells treated with retinoic acid (2.0×10^–5M) differentiate and acquire processes similar to neurons. Differentiated and logarithmically growing undifferentiated cells were exposed to varying doses of doxorubicin (an anthracycline chemotherapeutic antibiotic). While doxorubicin was lethal to many undifferentiated dividing cells, it was not as damaging to differentiated cells. After 2 to 4 days of doxorubicin treatment, the cells were harvested, the protein concentration determined and SDS-PAGE performed. Proteins were blotted onto nitrocellulose paper and immunostained with either a rabbit antiserum or mouse monoclonal antibody to tau. Undifferentiated LAN-5 cells treated with 4.0×10^–8M doxorubicin for 4 days and cells treated with 8.0×10^–8M doxorubicin for 2 days displayed a distinct lower band (just below the 50kd marker) that was either absent or very faint in untreated controls.Special issue dedicated to Dr. Paola S. Timiras.     

Role of SR-4987 stromal cells in the modulation of doxorubicin toxicity to in vitro granulocyte-macrophage progenitors (CFU-GM).

Bone marrow stromal microenvironment is essential for the maintenance of the hematopoietic stem cell renewal both by cell-cell interaction and cytokine production. However, stromal cells also exhibit drug metabolizing activities and they may accumulate the drug and successively affect hematopoietic progenitors by a retarded release. Our study investigated the role of both primary culture of murine bone marrow stroma and established stromal cells (SR-4987) in modulating the "in vitro" toxic activity of Doxorubicin (DXR) against murine granulocyte-macrophage progenitors (CFU-GM). The main part of the study has been performed by a "in vitro" agar bilayer technique based on the CFU-GM assay performed over a feederlayer of stromal cells. The results suggest that bone marrow stromal cells play also an important role in decreasing the toxicity of Doxorubicin. Further SR-4987 stromal cells produce a Doxorubicin metabolite (not belonging to the series of metabolites described in literature) which is completely ineffective in inhibiting the growth of CFU-GM and the activity of topoisomerase I. Our data suggest that bone marrow stromal cells must be considered as a cell population having opposite pharmacological roles in modulating the drug toxicity on hematopoietic progenitors. In our model a mechanism of detoxification concerns the capacity of SR-4987 stromal cells to inactivate the drug. For a better prediction of drug hematotoxicity, it is very important to develop "in vitro" cell models able to discriminate between positive and negative modulation of drug toxicity that stromal cells can exert in the bone marrow microenvironment.     

Inhibition of carbonic anhydrase activity modifies the toxicity of doxorubicin and melphalan in tumour cells in vitro

Carbonic anhydrase IX (CA IX) is a hypoxia-regulated enzyme, overexpressed in many types of human cancer. CA IX is involved in pH homeostasis, contributing to extracellular acidification and tumourigenesis. Acidification of the extracellular milieu can impact upon cellular uptake of chemotherapeutic drugs by favouring weak acids (e.g. melphalan), but limiting access of weak bases (e.g. doxorubicin). We investigated whether alterations of CA IX activity affected anti-cancer drug uptake and toxicity. CA inhibitor acetazolamide (AZM) enhanced doxorubicin toxicity but reduced melphalan toxicity in cell lines that highly expressed CA IX under anoxic conditions (HT29 and MDA435 CA9/18). The toxicity changes reflected modification of passive drug uptake. AZM did not alter toxicity or uptake in cells with low CA IX activity (HCT116 and MDA435 EV1). AZM lowered intracellular pH in HT29 and MDA435 CA9/18 cells under anoxic conditions. CA IX activity has chemomodulatory properties and is an attractive target for anti-cancer therapy.     

A switching mechanism in doxorubicin bioactivation can be exploited to control doxorubicin toxicity

Although doxorubicin toxicity in cancer cells is multifactorial, the enzymatic bioactivation of the drug can significantly contribute to its cytotoxicity. Previous research has identified most of the components that comprise the doxorubicin bioactivation network; however, adaptation of the network to changes in doxorubicin treatment or to patient-specific changes in network components is much less understood. To investigate the properties of the coupled reduction/oxidation reactions of the doxorubicin bioactivation network, we analyzed metabolic differences between two patient-derived acute lymphoblastic leukemia (ALL) cell lines exhibiting varied doxorubicin sensitivities. We developed computational models that accurately predicted doxorubicin bioactivation in both ALL cell lines at high and low doxorubicin concentrations. Oxygen-dependent redox cycling promoted superoxide accumulation while NADPH-dependent reductive conversion promoted semiquinone doxorubicin. This fundamental switch in control is observed between doxorubicin sensitive and insensitive ALL cells and between high and low doxorubicin concentrations. We demonstrate that pharmacological intervention strategies can be employed to either enhance or impede doxorubicin cytotoxicity in ALL cells due to the switching that occurs between oxygen-dependent superoxide generation and NADPH-dependent doxorubicin semiquinone formation.     

Effects of doxorubicin on human dental pulp cells in vitro

There is substantial information concerning the effects of continuous exposure to supratherapeutic or therapeutic concentrations of doxorubicin on human molar pulpal cells; the effects of continuous exposure to subtherapeutic concentrations of this agent are undetermined. To this end, we studied the proliferation of human fibroblasts and pulpal cells and their pattern of mineralized nodule deposition in vitro. Cell proliferation was assessed at 1, 3, 5, and 7 days from populations with either no exposure (control) or exposure to 10⁻⁶–10⁻⁹ mol/L doxorubicin. Mineralized nodule deposition and calcium-45 incorporation were assessed at 7 and 21 days of culture. Data were compared by factorial ANOVA and a post-hoc Tukey test. 10⁻⁶ and 10⁻⁷ mol/L doxorubicin significantly reduced the total number of viable pulpal cells in cultures from days 1 to 3 (p < 0.05); doxorubicin 10⁻⁶–10⁻⁹ mol/L significantly inhibited cell proliferation (p < 0.05) and DNA synthesis 5 days after plating (p < 0.001). After 21 days, doxorubicin 10⁻⁶–10⁻⁸ mol/L significantly decreased calcium-45 incorporation into pulpal cultures (p < 0.001); all dilutions significantly reduced the number of mineralized nodules within the 21-day pulpal cultures (p < 0.05). In addition, all dilutions of doxorubicin significantly inhibited fibroblast cell proliferation and incorporation of [³H]thymidine. In contrast, the fibroblast cultures did not produce mineralized nodules, suggesting that the mineralized nodules within the pulpal cell cultures did not result from dystrophic calcification. Thus, exposure to subtheraputic doxorubicin concentrations has potential adverse effects on mineralized tissue formation within the pulp, which could affect the rates of reparative dentin deposition within the tooth pulps of patients receiving this chemotherapeutic agent.     

Modulation of macrophage-mediated toxicity to cancer cells in vitro by liposomes and endotoxins

The cytotoxic effect of rat peritoneal macrophages on syngeneic colon cancer cells is modified by the addition of liposomes. The direction and intensity of this modification depends on the concentration of endotoxin in the culture medium.     

In vitro cytotoxic study of immunoliposomal doxorubicin targeted to human CD34(+) leukemic cells

The expression of CD34 antigen in acute myelogenous leukemias is considered an unfavourable prognosis marker for response to anticancer drugs and duration of remission. This study investigated the applicability of long-circulating immunoliposomes loaded with doxorubicin targeted to CD34 antigen present on MDR(+) human myelogenous leukemia KG-1a cell line. Immunoliposomal doxorubicin showed a higher cytotoxicity against KG-1a cells than non-targeted liposomal doxorubicin, but it did not improve over that of free drug. Although no reversal of doxorubicin resistance was found to occur through its liposomal encapsulation, a therapeutic benefit can be obtained by the selective cytotoxicity observed. Endocytosis studies demonstrated that, after binding to CD34 antigen, the immunoliposomes are not internalized by the KG-1a cells and so the cytotoxic effect might be due to drug released into the space near the cell membrane. Thus, immunotargeting of liposomal doxorubicin to CD34(+) leukemic cells may only provide an ex vivo strategy for site-selective CD34(+) leukemia cell killing.     

RNA/nucleotide enhances antibody production in vitro and is moderately mitogenic to murine spleen lymphocytes.

Suppression of immune functions was demonstrated in both humans and animals when exogenous RNA was eliminated from the diet. However, direct actions of RNA/nucleotide on the immune system are virtually unknown. Thus, in this study, we explored effects of RNA and nucleotide on lymphocyte functions in vitro. Yeast whole RNA, which is free of endotoxin, was supplemented to culture media, and changes in mitogen responses, thymocyte proliferation, or in vitro antibody production by murine spleen lymphocytes were analyzed. Yeast whole RNA potentiated the proliferation of spleen lymphocytes and it also strikingly enhanced in vitro antibody production in response to sheep red blood cells at least 10-fold. However, it did not potentiate the proliferation of thymocytes (immature lymphocytes). These enhancing activities of yeast RNA were significantly reduced by RNAse treatment, but not by treatments with DNAse or polymyxin B. Certain mononucleotides exhibited less, but similar, action on murine spleen lymphocytes. The whole yeast RNA employed was already degraded to small nucleotide (less than 1 kb). Therefore, it may be suggested that certain components of RNA degraders can function as powerful immunomodulators, indicating that exogenous RNA or nucleotide may be important in facilitating immune responses under certain circumstances.     

Gadopentatic acid affects in vitro proliferation and doxorubicin response in human breast adenocarcinoma cells

Contrasting agents (CAs) that are administered to patients during magnetic resonance imaging to facilitate tumor identification are generally considered harmless. However, gadolinium (Gd) based contrast agents can be retained in the body, inflicting specific cell line cytotoxicity. We investigate the effect of Gadopentatic acid (Gd-DTPA) on human breast adenocarcinoma MCF-7 cells. These cells exhibit a toggle switch response: exposure to 0.1 and 1 mM concentrations of Gd-DTPA enhances proliferation, which is hindered at a higher 10 mM concentration. Proliferation is enhanced when cells transition to 3D morphologies in post confluent conditions. The proliferation dependence on the concentration of CA is absent for Hs 578T and MDA-MB-231 triple negative cell lines. MCF-7 cells reveal a double toggle switch related to the expression of VEGF, which goes through high–low–high downregulation when cells are exposed to 0.1, 1, and 10 mM Gd-DTPA, respectively. Finally, doxorubicin drug response is assessed, which also reveals a double toggle switch behavior, where drug cytotoxicity exhibits a nonlinear dependence on the CA concentration. A toggle switch in cell characteristics that are exposed to 1 mM of Gd-DTPA amplifies the importance of this threshold, affecting several cell behaviors if surpassed. This work emphasizes the important effects that CAs can have on cells, specifically Gd-DTPA on MCF-7 cells, and the implications for cell growth and drug response during clinical and synthetic biology procedures.     

Copper toxicity to five Parmelia lichens in vitro

Treatment of Parmelia caperata, P. perlata, P. subrudecta, P. sulcata and P. tiliacea with CuSO₄ resulted in a time- and copper-concentration-dependent decrease in the total and intracellular potassium concentrations of the thallus, indicating that copper damaged the cytoplasmic membrane. Treatment with copper also resulted in a time-dependent increase in the total copper concentration of the thallus. After 4 h of exposure to copper, the process of potassium efflux was essentially completed but the absorption of copper was still increasing; moreover, the amount of copper bound to the thallus exceeded twice the amount of potassium released from the thallus, suggesting that cupric ions reached intracellular sites in the thallus, and K⁺/Cu²⁺ exchange was not electroneutral. After 5 h of exposure to copper, the extent of decrease in the total and intracellular potassium concentrations of the thallus was positively correlated with copper absorption levels, but only at 0.05<P<0.10, suggesting that membrane damage was proportional to the amount of bound copper, but other factors could have been operative, namely binding of copper to the cell wall. Acetone extracts of untreated thalli contained low concentrations of amino acids, polyols, and sugars, but considerable amounts of lichen substances: atranorin, caperatic, constictic, lecanoric, menegazziaic, protocetraric, salazinic, stictic, and usnic acids. Titration of the extracts with copper and assay of the free Cu²⁺ concentration revealed the presence of copper-binding ligands, and several successive absorption cycles, most probably corresponding to the binding of Cu²⁺ to each of the lichen substances detected in the extracts. However, no significant correlation (P>0.10) was found between the Cu²⁺-complexing capacity of acetone extracts and copper-induced membrane damage. It was concluded that in the studied Parmelia species, and in the experimental conditions used in this work, copper toxicity was not a simple function of the Cu²⁺-binding properties of the lichen substances present in the thallus. Several hypotheses were formulated to interpret the results.     

Quiescent hepatic stellate cells induce toxicity and sensitivity to doxorubicin in cancer cells through a caspase-independent cell death pathway: Central role of apoptosis-inducing factor

Hepatocellular carcinoma (HCC) is a major health problem worldwide and in the United States as its incidence has increased substantially within the past two decades. HCC therapy remains a challenge, primarily due to underlying liver disorders such as cirrhosis that determines treatment approach and efficacy. Activated hepatic stellate cells (A-HSCs) are the key cell types involved in hepatic fibrosis/cirrhosis. A-HSCs are important constituents of HCC tumor microenvironment (TME) and support tumor growth, chemotherapy resistance, cancer cell migration, and escaping immune surveillance. This makes A-HSCs an important therapeutic target in hepatic fibrosis/cirrhosis as well as in HCC. Although many studies have reported the role of A-HSCs in cancer generation and investigated the therapeutic potential of A-HSCs reversion in cancer arrest, not much is known about inactivated or quiescent HSCs (Q-HSCs) in cancer growth or arrest. Here we report that Q-HSCs resist cancer cell growth by inducing cytotoxicity and enhancing chemotherapy sensitivity. We observed that the conditioned media from Q-HSCs (Q-HSCCM) induces cancer cell death through a caspase-independent mechanism that involves an increase in apoptosis-inducing factor expression, nuclear localization, DNA fragmentation, and cell death. We further observed that Q-HSCCM enhanced the efficiency of doxorubicin, as measured by cell viability assay. Exosomes present in the conditioned media were not involved in the mechanism, which suggests the role of other factors (proteins, metabolites, or microRNA) secreted by the cells. Identification and characterization of these factors are important in the development of effective HCC therapy.     

Luteolin as a glycolysis inhibitor offers superior efficacy and lesser toxicity of doxorubicin in breast cancer cells

Luteolin (Lu) exhibits a wide spectrum of anti-tumor activities, the present study was to observe whether Lu can sensitize breast cancer cells to doxorubicin (Dox) and to explain the basis underlying this phenomenon. In vitro, Lu at dose less than 100 μM had only slight effect on cells growth and cytotoxicity of Dox in 4T1 and MCF-7 cells under normoxia, but it could reverse tumor resistance to Dox and promote death of tumor cells under hypoxia. In vivo, Lu alone had also no effect on tumor growth delay, however, it could offer superior efficacy and lesser toxicity of Dox in 4T1 and MCF-7 bearing mice. Further study showed that Lu was able to suppress glycolytic flux but did not affect glucose uptake, the P-glycoprotein, anti-oxidative enzymes under hypoxia in vitro, and had not also effect on the intratumor Dox level in vivo. In addition, the activity of SOD and CAT was increased in serum and was decreased in tumor by Lu in vivo. These results suggest that luteolin as a glycolytic inhibitor might be a new adjuvant agent for chemotherapy.     

Inflammatory microcrystals alter the functional phenotype of human osteoblast-like cells in vitro: synergism with IL-1 to overexpress cyclooxygenase-2

Chronic crystal-associated arthropathies such as gout and pseudogout can lead to local bone destruction. Because osteoblasts, which orchestrate bone remodeling via soluble factors and cell-to-cell interactions, have been described in contact with microcrystals, particularly in uratic foci of gout, we hypothesized that microcrystals of monosodium urate monohydrate (MSUM) and of calcium pyrophosphate dihydrate (CPPD) could alter osteoblastic functions. MSUM and CPPD adhered to human osteoblastic cells (hOB) in vitro and were partly phagocytized as shown by scanning electron microscopy. MSUM and CPPD dose-dependently stimulated the production of PGE(2) in hOB as assessed by enzyme immunoassay, a response that was synergistically enhanced in the presence of IL-1. The mechanism of this synergism was, at least in part, at the level of the expression of cyclooxygenase-2 as evaluated by immunoblot analysis. MSUM and CPPD also stimulated the expression of IL-6 and IL-8 and reduced the 1,25-dihydroxyvitamin D(3)-induced activity of alkaline phosphatase and osteocalcin in hOB (with no synergism with IL-1). MSUM- or CPPD-stimulated expression of IL-6 in hOB pretreated with the selective cyclooxygenase-2 inhibitor NS-398 was increased, unlike that induced by IL-1 alone which was partially reduced. MSUM-, CPPD- or IL-1-induced expression of IL-8 was unchanged by pretreating hOB with NS-398. These results suggest that inflammatory microcrystals alter the normal phenotype of hOB, redirecting them toward reduced bone formation and amplified osteoblast-mediated bone resorption, abnormalities that could play a role in the bone destruction associated with chronic crystal-induced arthritis.     

Hepatocytes--the choice to investigate drug metabolism and toxicity in man: in vitro variability as a reflection of in vivo

The pharmaceutical industry is committed to marketing safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. Drug metabolism is a major determinant of drug clearance and interindividual pharmacokinetic differences, and an indirect determinant of the clinical efficacy and toxicity of drugs. Progressive advances in the knowledge of metabolic routes and enzymes responsible for drug biotransformation have contributed to understanding the great metabolic variations existing in human beings. Phenotypic as well genotypic differences in the expression of the enzymes involved in drug metabolism are the main causes of this variability. However, only a minor part of phenotypic variability in man is attributable to gene polymorphisms, thus making the definition of a normal liver complex. At present, the use of human in vitro hepatic models at early preclinical stages means that the process of selecting drug candidates is becoming much more rational. Cultured human hepatocytes are considered to be the closest model to human liver. However, the fact that hepatocytes are located in a microenvironment that differs from that of the cell in the liver raises the question: to what extent does drug metabolism variability observed in vitro actually reflect that of the liver in vivo? By comparing the metabolism of a model compound both in vitro and in vivo in the same individual, a good correlation between the in vitro and in vivo relative abundance of oxidized metabolites and the hydrolysis of the compound was observed. Thus, it is reasonable to consider that the variability observed in human hepatocytes reflects the existing phenotypic heterogeneity of the P450 expression in human liver.     

In vitro toxicity of T-2 mycotoxin in mouse lymphoid cells

The in vitro toxicity of T-2 toxin towards mouse lymphoid cells prepared from spleen, thymus, peritoneal lavage and bone marrow cells was studied. Bone marrow cells were more resistant to damage by T-2 toxin than thymus, spleen and peritoneal cell preparations.