Doxorubicin and epirubicin iron-induced generation of free radicalsIn vitro. A comparative study

To ascertain any differences in myocardial injury exerted by the anthracyclines doxorubicin and epirubicin, their ability to generate oxygen free radicals when mixed with Fe(II) was examined in vitro using an oxygen electrode. 5–250 g/ml doxorubicin or epirubicin consumed oxygen when mixed with 50 or 100 mol/1 Fe(II). Addition of 75 mol/1 cytochrome C showed that of the consumed oxygen, approximately 80% entered the monovalent pathway of oxygen reduction. The strong inhibitory effect of 250 mg/1 catalase indicates that most of the superoxide radicals generated are further reduced to hydrogen peroxide by both anthracyclines. Addition of metal chelators DTPA (100/mol/1), or DDTC (50 mol/1) did not affect oxygen consumption, whereas EDTA (100/mol/1) or desferrioxamine (100 mol/1) with anthracyclines and Fe(II) rather stimulated oxygen consumption. It is concluded that there are no significant differences in the amount or proportion of generated oxygen free radicals between doxorubicin and epirubicin when mixed with Fe(II) in a cell-free system in vitro. Thus, the ability of the anthracyclines, in conjunction with iron alone, to generate radicals does not explain the differences of the drugs in causing myocardial injury.     

P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines

MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings.     

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.     

Copper deficient rat heart can compensate for doxorubicin-induced oxidant stress

The hypothesis that copper (Cu) alters drug metabolizing enzymes and functions as an antioxidant nutrient in doxorubicin cardiotoxicity was tested. Male Sprague-Dawley rats were fed Cu adequate (⁺Cu; 5 mg Cu/kg of diet), marginally Cu deficient (MCu; 1.2 mg Cu/kg of diet), or severely Cu deficient (⁻Cu; 0.5 mg Cu/kg of diet) diets for 6 wk. Doxorubicin (1, 2, or 4 mg/kg body wt) or saline were administered intraperitoneally 1 time/wk for 4 wk. Compared to control hearts, Cu, Zn superoxide dismutase activity was decreased by 9% in MCu rats and by 21–40% in⁻Cu rats. Glutathione peroxidase activity was elevated 5–15% in⁻Cu rats. Doxorubicin administration increased heart Cu, Zn superoxide dismutase activity in⁺Cu and⁻Cu rats 18 h after the last of 4 injections, but not 18 h after 1 injection. There was no synergism between doxorubicin and Cu deficiency on lipid peroxidation, plasma creatine phosphokinase, cardiac hypertrophy, electrocardiographic abnormalities, or morphological changes. Heart glutathione S-transferase activity was decreased by Cu deficiency, and like Cu, Zn superoxide dismutase activity, returned to normal in⁻Cu rats given doxorubicin. Thus, the Cu deficient rat heart may be able to compensate for doxorubicin-induced oxidant stress by increasing the activity of Cu,Zn superoxide dismutase and glutathione S-transferase.     

Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses

Summary— Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.     

Nimbolide prevents myocardial damage by regulating cardiac biomarkers,antioxidant level,and apoptosis signaling against doxorubicin‐induced cardiotoxicity in rats

The current work planned to assess the protecting properties of nimbolide against doxorubicin (DOX)‐treated myocardial damage. Myocardial damage was produced with 2.5 mg/kg of DOX given on alternative days (14 days). Thiobarbituric acid reactive substances (TBARS) levels of a lipid peroxidative marker were elevated, whereas reduced body weight, heart weight, blood pressure indices and reduced levels of antioxidants like glutathione‐S‐transferase, superoxide dismutase, catalase, glutathione peroxidase, glutathione, and glutathione reductase were observed in the heart tissue of DOX‐treated animals. DOX‐treated animals showed augmented levels of cardiac markers likes monocyte chemotactic protein‐1, interferon‐gamma, aspartate transferase, creatine kinase, lactate dehydrogenase, creatine kinase‐muscle/brain, heart‐type fatty acid‐binding protein, glycogen phosphorylase isoenzyme BB, transforming growth factor‐β, brain natriuretic peptide, myoglobin, and cTnI in serum. Histopathological assessment confirmed the DOX‐induced cardiotoxicity. Furthermore, DOX‐induced rats showed augmented inflammatory mediators (nuclear factor‐κB [NF‐kB], tumor necrosis factor‐α [TNF‐α], and interleukin‐1β [IL‐1β]) and increased PI3K/Akt signaling proteins (PI3K, p‐Bad/Bad, caspase‐3, and p‐Akt), whereas decreased oxidative markers (HO‐1 and NQO‐1) and p‐PTEN were observed. Nimbolide‐supplemented rats showed reduced activity/levels of cardiac markers and TBARS levels in serum and heart tissue. Levels of enzymatic and nonenzymatic antioxidants were augmented in the heart tissue of nimbolide‐supplemented rats. Nimbolide influence decreased apoptosis, inflammation, and enhanced antioxidant markers through the modulation of p‐Bad/Bad, caspase‐3, PI3K, p‐Akt, TNF‐α, NF‐kB, IL‐1β, HO‐1, NQO‐1, and p‐PTEN markers. The histopathological explanations were observed to be in line with biochemical analysis. Therefore, the finding of current work was that nimbolide has a defensive effect on the myocardium against DOX‐induced cardiac tissue damage.     

Polydatin ameliorates chemotherapy-induced cognitive impairment (chemobrain) by inhibiting oxidative stress,inflammatory response,and apoptosis in rats

ABSTRACT

Most breast cancer survivors receiving chemotherapy have severe cognitive impairment, often referred to as “chemobrain.” Polydatin (PLD) is known to have many biological activities. Thus, this study aimed to determine whether symptoms of chemobrain can be prevented or relieved by PLD. The chemobrain models were established by intraperitoneal injection of doxorubicin (DOX, 2 mg/kg) in rats once a week for 4 weeks (DOX group and DOX+PLD group). In the PLD group and DOX+PLD group, PLD (50 mg/kg) was administered orally to rats every day. We found that PLD treatment significantly protected against DOX-induced learning and memory impairment, restored hippocampal histopathological architecture. Furthermore, PLD suppressed DOX-induced oxidative stress through up-regulating Nrf2, inhibited inflammatory response by activating the NF-κB pathway, and reduced hippocampal apoptosis. Therefore, the present study indicated that PLD offered neuroprotection against DOX-induced chemobrain. PLD may assist in preventing chemobrain after chemotherapy in patients with cancers.     

Thymoquinone attenuates doxorubicin‐cardiotoxicity in rats

Contrary to the fact that doxorubicin is a powerful chemotherapeutic agent for the treatment of neoplastic diseases, cardiotoxicity is too important to be ignored. Thymoquinone serves as a powerful free radical scavenger. In the study, the effects of thymoquinone against doxorubicin‐cardiotoxicity will be evaluated. Forty rats were divided into five groups. Group I: control group (n = 8); group II: olive oil group (n = 8); group III: thymoquinone group (n = 8); given 10 mg/kg thymoquinone intraperitoneally per day throughout the experiment; group IV: doxorubicin group (n = 8); injected with a single dose of 15 mg/kg ip doxorubicin on the 7th day of the experiment; group V: doxorubicin + thymoquinone group (n = 8); administered with 10 mg/kg thymoquinone per day during the experiment and 15 mg/kg doxorubicin ip on the 7th day. The experiment was planned for 14 days. Immunohistochemically, heat shock protein (HSP) 70 and HSP90, glucose‐regulated protein 78 (GRP78), caspase‐3 were stained. We made terminal deoxynucleotidyl transferase dUTP nick end labeling for apoptotic evaluation. Total oxidant status (TOS) levels and total antioxidant status (TAS) were measured in the heart tissue. Atrial natriuretic peptide (ANP) and pro‐B type natriuretic peptide (proBNP) were evaluated. In the study, HSP70, HSP90, GRP78, and caspase‐3 levels increased in group IV. TOS and TAS levels were significant compared to group I. Doxorubicin significantly increased ANP and NT‐proBNP levels. Thymoquinone revealed significant differences in these values. Thymoquinone can be an important cardioprotective agent against doxorubicin‐cardiotoxicity.     

Redox Cycling in MCF-7 Human Breast Cancer Cells by Antitumor Agents Based on Mitozantrone

In a series of hydroxyethylaminoalkylaminoanthraquinones (AQ's) based on mitozantrone, 1-AQ (340%) and 1,8-AQ (137%) stimulated basal rate NADPH oxidation (72 + 18pmol min^-lmg S9 protein^-1) whilst 1,4-AQ, 1,5-AQ and mitozantrone had no effect. A similar trend was observed for O_2? generation (measured as nmol acet. cyt c reduction min-¹ mg protein^-1) by these compounds in MCF-7 S9 fraction: 1-AQ (9.5) and 1,8-AQ (7.9), whilst 1,5-AQ, 1,4-AQ and mitozantrone showed no significant effect. All the AQs including mitozantrone were cytotoxic to MCF-7 cells in a dose dependent manner with EC₅₀ values as follows: 1-AQ (0.01 μm) > doxorubicin (0.4μM) > mitozantrone (0.6μM) > 1,8-AQ (2.O μM) > 1,5-AQ (4.0μM) > 1,4-AQ (8,0 μM). Thus the redox active AQs were also the most cytotoxic. Mitozantrone however was not redox active but was more cytotoxic than all but 1-AQ hence it would appear that factors other than free radical generation contribute to the antitumor activity of this group of compounds.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.