Cytotoxic Effects Of Adriamycin Research Articles

Assessment of the Nutraceutical Effects of Oleuropein and the Cytotoxic Effects of Adriamycin, When Administered Alone and in Combination, in MG-63 Human Osteosarcoma Cells.

Oleuropein (OLEU) is the most distinguished phenolic compound found in olive fruit and the leaves of Olea europaea L., with several pharmacological properties, including anti-cancer actions. Adriamycin (ADR) is an anthracycline widely used as a chemotherapeutic agent, although it presents significant side effects. The aim of the present study was to investigate the effect of oleuropein alone (20 μg/mL) and in co-treatment with ADR (50 nM), in MG-63 human osteosarcoma cells. Therefore, cellular and molecular techniques, such as MTT assay, flow cytometry, real-time Polymerase Chain Reaction (PCR), western blot and Elisa method, as well as Nuclear Magnetic Resonance (NMR) spectroscopy, were applied to unveil changes in the signal transduction pathways involved in osteosarcoma cells survival. The observed alterations in gene, protein and metabolite levels denote that OLEU not only inhibits MG-63 cells proliferation and potentiates ADR’s cytotoxicity, but also exerts its action, at least in part, through the induction of autophagy.

Effect of gap junction modulation on antitumor effects of adriamycin in estrogen receptor-positive breast cancer cells

To observe the effect of functional modulation of gap junctions (GJ) on the antitumor effect of adriamycin in breast cancer cells positive for estrogen receptor (ER). The inhibitory effect of 0 to 24.0 μmol/L adriamycin on the surviving fraction of ER-positive human breast cancer MCF-7 cells and ER-negative MDA-MB-231 cells was assessed with MTT assay; Western blotting and immunofluorescence assay were used to detect the expressions of Cx43 total protein and membrane protein in the cells. A parachute assay was used to evaluate the function of the GJ in MCF-7 cells. The cytotoxic effect of adriamycin was observed in the cells treated with retinoic acid (RA) for enhancing GJ function, in cells treated with oleamide and 18-α- glycyrrhizic acid (18-α-ga) for inhibiting GJ function, and also in cells transfected with Cx43siRNA for Cx43 knockdown. ER-positive MCF-7 cells expressed a significantly higher level of Cx43 with stronger GJ function than ER-negative MDA- MB-231 cells. Adriamycin significantly inhibited the proliferation of MCF-7 cells (P < 0.01), and RA treatment further increased the cytotoxicity of adriamycin (P < 0.01) while oleamide and 18-α-GA obviously attenuated the cytotoxicity of adriamycin (P < 0.01). In the cells with Cx43 knockdown, the expressions of total Cx43 protein and Cx43 on the membrane were significantly reduced, the function of GJ was attenuated, and the cytotoxicity of adriamycin was significantly decreased (P < 0.01). ER-positive breast cancer cells have stronger Cx43 expressions and GJ function than the ERnegative cells. The cytotoxicity of adriamycin against the breast cancer cells can be strengthened by enhancing GJ function and attenuated by inhibiting GJ function. Cx43 silencing inhibits the function of GJ to lower the cytotoxicity of adriamycin in human breast cancer MCF-7 cells.

Naringenin suppresses K562 human leukemia cell proliferation and ameliorates Adriamycin-induced oxidative damage in polymorphonuclear leukocytes.

Treatments for leukemia remain unsatisfactory. Conventional chemotherapy agents that aim to kill tumor cells may also damage normal cells and thus result in severe side-effects. Naringenin, a natural polyphenolic compound with antioxidant effects, has been revealed to have significant antitumor effects with low toxicity in preliminary studies. Thus, it is considered as one of the most promising flavonoids in the treatment of leukemia. In the present study, the effects of naringenin on the K562 human leukemia cell line and the underlying mechanisms were explored in vitro. In addition, human peripheral blood polymorphonuclear leukocytes (PMNs) were used as a normal control in order to evaluate the effects of naringenin on normal granulocytes and in the mediation of Adriamycin (ADM)-induced oxidative damage. The results revealed that K562 proliferation was significantly inhibited by naringenin in a time- and concentration-dependent manner; however, minimal cytotoxic effects were observed in PMNs when naringenin was used at concentrations <400 μmol/l. Morphological changes indicative of apoptosis were observed in naringenin-treated K562 cells. Flow cytometric analysis indicated that the K562 cells were arrested in the G0/G1 phase of the cell cycle with a significantly upregulated rate of apoptosis. Furthermore, in the naringenin-treated K562 cells, the labeling index of proliferating cell nuclear antigen was observed to be increased by immunochemical staining, the mRNA and protein expression levels of p21/WAF1 were strongly upregulated in reverse transcription-polymerase chain reaction and western blot analyses, whereas p53 gene expression was not significantly changed. In PMNs to which naringenin (50~80 μmol/l) was added 1 h subsequent to ADM, the cell damage induced by ADM was significantly reduced, coincident with reductions in the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and increases in the activity of superoxide dismutase and glutathione peroxidase. However, the cytotoxic effect of ADM in K562 cells was not significantly altered by naringenin, and the oxidative stress indices in K562 cells remained stable. In conclusion, the present study revealed the promising value of naringenin in leukemia treatment. Naringenin demonstrated a significant inhibitory effect on the growth of K562 cells but not on normal PMNs. Furthermore, naringenin protected PMNs from ADM-induced oxidative damage at low concentrations. Cell cycle arrest and apoptosis-inducing effects, achieved through p53-independent p21/WAF1 upregulation, are likely to be the mechanism of the antileukemic effects of naringenin, and the protective effect against ADM chemotherapy-induced damage in PMNs may be due to the antioxidant capability of this agent at low concentrations.

Synergistic effect of hyperthermia and neferine on reverse multidrug resistance in adriamycin-resistant SGC7901/ADM gastric cancer cells

Multidrug resistance (MDR) plays a major obstacle to successful gastric cancer chemotherapy. The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine (Nef) in adriamycin (ADM) resistant human SGC7901/ADM gastric cancer cells. The MDR cells were heated at 42°C and 45°C for 30 min alone or combined with 10 μg/mL Nef. The cytotoxic effect of ADM was evaluated by MTT assay. Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique. Intracellular accumulation of ADM was monitored with high performance liquid chromatography. Mdr-1 mRNA, P-glycoprotein (P-gp), γH2AX expression and γH2AX foci formation were determined by real-time PCR, Western blot and immunocytochemical staining respectively. It was found that different heating methods induced different cytotoxic effects. Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells. The water submerged hyperthermia increased the cell membrane fluidity. Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM. The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp. The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells. The higher temperature was, the worse effect was. Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.

The Effect of β-Carotene Against Adriamycin Toxicity on the Embryo Formation

Adriamycin is an anthracycline antibiotic widely used for the treatment of many types of cancer. The cytotoxic effect of Adriamycin occurs by a free radical-mediated mechanism. Thus, to prevent or reduce the toxic effect of Adriamycin, it is possible to use it in combination with antioxidants. The aim of this study was to evaluate a potential effect of β-carotene against Adriamycin-induced toxicity on the embryo formation. Pregnant rats were treated with Adriamycin, β-carotene, and their combination during the critical stages of embryogenesis. The first group was control group. Adriamycin was administered on day 9 (group 2a) and day 12 (group 2b) of gestation by a single intraperitoneal injection at a dose of 5 mg/kg. β-Carotene was given at a dosage of 0.6 mg/(kg·day) from day 6 to 10 or from day 9 (group 3a) to 13 (group 3b) of gestation 5 times per os; in the case of their combination, β-carotene was given per os 3 times before Adriamycin injection, one time simultaneously with Adriamycin and one time after its injection (groups 4a and 4b). Animals were euthanized on day 21 of gestation. Embryo resorptions and alive fetuses were counted, weighed, and measured. The embryos of each litter were examined macroscopically after the Buen solution fixation for the embryo defects. In order to render the skeleton visible, the soft tissues were macerated using caustic soda, stained with alizarin red, and cleared with glycerin. Adriamycin induced embryotoxicity; the combination of Adriamycin and β-carotene decreased the number of Adriamycin-induced embryo resorptions about two times. A gavage with Adriamycin alone decreased fetal body weights (P<0.05), while giving it in combination, the fetal body weight was similar to that in the control group. Adriamycin induced the retardation of skeletogenesis and external fetal malformations (microphthalmia, hydrocephaly, anencephaly, and others). After an exposure to β-carotene, external malformations (diaphragmatic hernia) of embryos were found only occasionally. β-Carotene in combination with Adriamycin produced no positive effect on Adriamycin-induced skeletodysgenesis or external malformations. Antioxidant β-carotene in combination with Adriamycin slightly reduced the Adriamycin-induced embryotoxicity, but produced no positive effect on Adriamycin-induced skeleto-dysgenesis or external malformations.

Enhancing the Antitumor Activity of Adriamycin and Ionizing Radiation

Overexpression of manganese superoxide dismutase (MnSOD), when combined with certain chemicals that inhibit peroxide removal, increases cancer cell cytotoxicity. Elevating MnSOD levels in cells enhances the conversion of superoxide (O(2)(*-)) to hydrogen peroxide (H(2)O(2)), combined with inhibiting the removal of H(2)O(2), further increases H(2)O(2) levels, leading to increased cytotoxicity. We hypothesized that increasing endogenous O(2)(*-) production in cells that were pretreated with adenoviral MnSOD (AdMnSOD) plus 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) would lead to an increased level of intracellular H(2)O(2) accumulation and increased cell killing. The cytotoxic effects of Adriamycin or radiation, agents known to produce O(2)(*-), were determined in MDA-MB-231 breast cancer cells pretreated with AdMnSOD plus BCNU both in vitro and in vivo. In vitro, AdMnSOD plus BCNU sensitized cells to the cytotoxicity of Adriamycin or radiation. In vivo, AdMnSOD, BCNU, and Adriamycin or ionizing radiation inhibited tumor growth and prolonged survival. The results suggest that agents that produce O(2)(*-) in combination with AdMnSOD plus BCNU may represent a powerful new antitumor regimen against breast cancer.

Intracellular uptake, retention and cytotoxic effect of adriamycin combined with hyperthermia in vitro.

The intracellular uptake, retention and cytotoxic effect of adriamycin (ADR) were investigated by a flow cytometry technique with NIH 3T3 cells. The intracellular uptake and cytotoxic effect of ADR increased as a function of exposure time and external drug concentration at 37 degrees. A good correlation was found between eventual survival and intracellular ADR uptake. The intracellular uptake and cytotoxic effect of ADR increased with increasing temperature and were shown to be functions of both incubation time and temperature. The intracellular uptake and cytotoxic effect of ADR were increased even at 39 degrees and 41 degrees, temperatures which had no effect on the viability of cells. The combination of hyperthermia (43 degrees) and ADR showed a synergistic effect. It was found that the cytotoxic effects of ADR in combination with various levels of hyperthermia were stronger than those which would be predicted from the intracellular ADR uptake at 37 degrees. The degree of enhancement was temperature-dependent. Since the efflux of intracellular ADR was the same with or without hyperthermia, the increased intracellular ADR uptake caused by elevated temperature was considered to result from an increase in influx.

Potentiation by squalene of the cytotoxicity of anticancer agents against cultured mammalian cells and murine tumor.

Squalene (SQ) was examined for ability to potentiate the cytotoxicity and antitumor activity of anticancer agents. The dose-response curves of cell survival of cultured HeLa or V79 cells in the presence of various anticancer agents [adriamycin (ADM), 5-fluorouracil (5-FU), bleomycin (BLM) and cis-dichlorodiamminoplatinum (CDDP)] showed that SQ potentiated the cytotoxicity of these anticancer agents. SQ may enhance the cytotoxic effect of ADM by interfering with the efflux of ADM from the cells. The antitumor activities of these anticancer agents combined with SQ were tested against sarcoma 180 (S180) ascites cells. Some combinations were found to show synergistic antitumor effects: ADM, BLM or CDDP with SQ was effective against S180, but only slight if any synergistic effect was observed with the combination of 5-FU and SQ. The antitumor activity of BLM was most strongly potentiated by SQ, among the tested agents.

Erythropoietin Fails to Interfere with the Antiproliferative and Cytotoxic Effects of Antitumor Drugs

Erythropoietin (EPO) therapy is widely used for the prevention and treatment of anemia resulting from cancer chemotherapy. Native EPO regulates erythropoiesis, at least in part, by protecting erythroid progenitor cells from apoptotic cell death. The recent discovery of the EPO receptor (EPOR) on cancer cells raises the concern that EPO therapy might stimulate tumor growth and/or protect cancer cells from drug-induced apoptosis. Therefore, the capacity of EPO to interfere with the effects of conventional chemotherapeutic drugs on proliferation, apoptosis, and the induction of senescence was investigated in MCF-7 and MDA-MB231 breast tumor cells, which express the EPOR as well as in F-MEL erythroleukemia cells. Breast cancer cells and F-MEL leukemic cells were cultured in the presence or absence of EPO and then exposed to antitumor drugs. Cell proliferation was assessed by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction assay 72 hours after drug exposure. Cytotoxicity was monitored by clonogenic survival. Apoptosis was evaluated either by the terminal deoxyribonucleotide transferase-mediated nick-end labeling assay or fluorescence-activated cell sorting analysis, and senescence was monitored by beta-galactosidase staining. EPO signaling was assessed by monitoring the phosphorylation/activation of specific signaling proteins. EPO failed to stimulate the proliferation of MCF-7 or MDA-MB231 breast tumor cells or F-MEL leukemic cells. EPO treatment also failed to interfere with the antiproliferative and/or cytotoxic effects of Adriamycin, Taxol, and tamoxifen in breast tumor cells (or of cytarabine and daunorubicin in F-MEL cells). EPO failed to prevent apoptosis induced by Taxol or senescence induced by Adriamycin in MCF-7 cells. EPO stimulated the activation of extracellular signal-regulated kinase, p38, and c-Jun-NH(2)-kinase in MCF-7 cells but did not activate Akt or signal transducers and activators of transcription 5 (STAT5). EPO failed to activate any of these signaling pathways in MDA-MB231 cells. Cytarabine and daunorubicin interfered with EPO signaling in F-MEL cells. These findings suggest that EPO is unlikely to directly counteract the effectiveness of cancer chemotherapeutic drugs. This may be a consequence of either ineffective signaling through the EPOR or drug-mediated suppression of EPO signaling.

The vitamin D3 analog, ILX-23-7553, enhances the response to adriamycin and irradiation in MCF-7 breast tumor cells.

Ionizing radiation and the anthracycline antibiotic, Adriamycin, generally fail to promote a primary apoptotic response in experimental breast tumor cell lines. Similarly, the primary response of breast tumor cells to vitamin D3 (1,25(OH)2D3) and vitamin D3 analogs such as EB 1089 is growth inhibition. Previous studies have demonstrated that pretreatment of MCF-7 breast tumor cells with vitamin D3 or EB 1089 can increase sensitivity to both Adriamycin and irradiation. The capacity of the vitamin D3 analog, ILX 23-7553, to enhance the antiproliferative and cytotoxic effects of Adriamycin or irradiation and to promote apoptosis in MCF-7 breast tumor cells was assessed in the present study. Pretreatment of MCF-7 cells with ILX 23-7553 followed by Adriamycin or irradiation decreased viable cell numbers by 97% and 93%, respectively. Cell numbers were reduced by 56%, 74% and 75% by ILX 23-7553, Adriamycin and irradiation alone. Pretreatment with ILX 23-7553 also shifted the dose response curve for clonogenic survival, increasing sensitivity to Adriamycin 2.5-fold and sensitivity to radiation fourfold. In addition, ILX 23-7553 pretreatment conferred sensitivity to Adriamycin- or irradiation-induced DNA fragmentation and resulted in morphological changes indicative of apoptotic cell death in MCF-7 cells. Statistical analysis demonstrated that ILX 23-7553 interacts additively and not synergistically with both Adriamycin and irradiation. ILX 23-7553 enhances the effects of Adriamycin and irradiation in MCF-7 breast tumor cells by decreasing viable cell numbers, reducing clonogenic survival and inducing apoptotic cell death. Current studies are focused on elucidating the mechanisms underlying the induction of apoptosis as well as understanding the nature of the interactions between ILX 23-7553 and Adriamycin or irradiation.

Characterization and In Vitro Cytotoxic Effect of Adriamycin-conjugated Monoclonal Antibody Prepared Against Breast Cancer Cell Line.

We have produced an adriamycin-conjugated monoclonal antibody, AM1, covalently linked by acid-labile cis-aconitic spacer. The immunoconjugate, cis-aconitic adriamycin(cAA)-AM1, was confirmed to retain the binding activity against human breast cancer cell lines by flow cytometry. The immunoconjugate was shown to be internalized into antigen-positive cancer cells by flow cytometry analysis and high performance liquid chromatography. Antitumor effects of cAA-AM1 were assessed on human breast cancer and colon cancer cell lines, with inhibition of (3) H-leucine uptakes to the cells. cAA-AM1 demonstrated a selective cytotoxicity to ZR-75-1 which was reactive with AM1, whereas it showed no antitumor effect on SW1116 cells which did not react with AM1. Free adriamycin demonstrated a non-selective cytotoxicity against both cell lines. AM1 alone and cAA-control IgM did not show any antitumor effect on ZR-75-1. These results suggest that cAA-AM1 retains binding activity and specificity against breast cancer cells in vitro.

Effect of verapamil on adriamycin resistant bladder tumor cells

RBT-K5 cell line was established from a male Wistar rat bladder tumor induced with N-butyl-N-(4-hydroxybutyl) nitrosamine. RBT-K5 (ADM res.) cell line, adriamycin (ADM) resistant, was also established from RBT-K5 cells by stepwise escalation of ADM concentrations in vitro. In this study, we have examined the effect of verapamil (VR) on intracellular ADM concentration in these cell lines. The viability of RBT-K5 cells was significantly lower than that of RBT-K5 (ADM res.) at the concentration from 0.5 to 4 micrograms/ml of ADM. The cytotoxic effect of ADM was enhanced by the addition of VR in the both cell lines. The intracellular ADM concentrations of RBT-K5 and RBT-K5 (ADM res.) were increased from 607.7 +/- 74.1 ng/10(6) cells to 1152.1 +/- 187.6 ng/10(6) cells (1.9-fold) and from 185.1 +/- 36.5 ng/10(6) cells to 751.5 +/- 258.4 ng/10(6) cells (4.1-fold), respectively, 24 hours after incubation in the presence of 10 micrograms/ml of VR. Furthermore, the intracellular ADM concentration was maintained at high-level in combination with VR even after the removal of ADM from culture medium. These results suggest that the combination therapy with VR is more effective against ADM-resistant than ADM-sensitive bladder tumor.

In vitro potentiation by lonidamine of the cytotoxic effect of adriamycin on primary and established breast cancer cell lines.

Lonidamine is a new potential chemotherapeutic agent, relatively non-toxic, that can positively modulate the efficacy of several antineoplastic drugs. We evaluated the response of two established human breast cancer cell lines (MCF-7 and BRC-230) and of 20 primary breast cancer cell lines to lonidamine, either alone or in combination with adriamycin, the drug most widely used in the management of breast cancer. Different schedules were tested by varying either concentration of the drugs (LND: 10-150 micrograms/ml; ADM: 0.10-0.15 micrograms/ml), or time of exposure (1-96 hours), or sequence of administration (ADM-->LND; LND-->ADM; ADM+LND). Our results indicate slight sensitivity of the cell lines to lonidamine when used alone, whereas an increase of efficacy was noted when lonidamine was added for at least 24 hours after a 4 hour exposure to adriamycin. Such efficacy was significantly greater than that expected from an additive effect between the two drugs. We conclude that lonidamine, when given according to an appropriate schedule, enhances, in vitro, the efficacy of adriamycin. A correct employment of lonidamine in the management of breast cancer might therefore potentiate the therapeutic effect of adriamycin.

Cytotoxic effect of adriamycin and agarose-coupled adriamycin on glomerular epithelial cells: Role of free radicals

It has been suggested that the generation of toxic radicals plays an important role in toxicity by Adriamycin (ADR) on cancer cell lines and in vivo. We have examined the role of free radicals in determining toxicity and resistance to ADR of rat glomerular epithelial cells in culture; this method provides a good model for analyzing the mechanisms responsible for ADR experimental nephrosis in rats. Three points were established: a) the intra- or extracellular site of ADR toxicity; b) the role of the superoxide anion and of the hydroxyl radical in determining intra- and -extracellular cytotoxicity; and c) the implication of oxido-reduction cycling as a potential route for ADR semiquinone transformation. Free ADR was found to induce the same inhibition of [3H]thymidine incorporation into DNA as ADR bound to an agarose macroporous bed which prevents the intracellular incorporation of the drug. Specific scavenging of free radical activity by the enzymes catalase and superoxide dismutase, the hydroxyl radical inhibitors dimethyl sulfoxide and dimethylthiourea (DMTU) and by chelation of intracellular free iron with deferoxamine produced only a partial restoration of [3H]thymidine incorporation into DNA, which was maximal for DMTU (30% of normal incorporation). DMTU treatment was unsuccessful in preventing the extracellular cytostatic effect of ADR. Finally, glomerular epithelial cell killing (51Cr-release method) by 5-iminodaunorubicin, an ADR analogue with a modified quinone function that prohibits oxido-reduction cycling, was higher than unmodified ADR. These results indicate that ADR may exert its cytotoxic effects on glomerular epithelial cells by interaction at the cell surface, whereas the intracellular compartment, principally DNA, does not seen to be the target of ADR effects. They also suggest that the free radicals are in part responsible for ADR intracellular cytotoxicity, but other mechanisms should also be hypothesized. Finally, the participation of the ADR semiquinone radical in oxido-reduction cycling seems not important for the induction of the cellular damage.

Sensitivity to thermochemotherapy of AKR lymphoma and B16 melanoma variants of malignancy

Drug resistance, which so often accompanies tumor progression, has been shown to be related to changes in membrane properties which may result in decreased drug accumulation in the tumor cell. A correlation between sensitivity to thermochemotherapy and degree of malignancy was found in the AKR lymphoma system. Hyperthermia increased adriamycin (ADR) uptake and concomitantly its cytotoxicity to AKR lymphoma cells. Moreover, these effects were more pronounced on a variant of high malignancy (HM) than on a low malignancy (LM) one. Fluorescent microscopy, as well as cytofluorometry, indicated that lymphoma cells treated by ADR at 43 degrees C were more permeable to the cytotoxic agent than those exposed to the chemotherapeutic substance at 37 degrees C. Cytofluorometry indicated the presence of a minor cell subpopulation with low ADR uptake in the HM variant, not found in the LM one. Fluorocytometry also showed that the temperature-dependent increased ADR uptake was more marked in the HM than in the LM variant, explaining the differential effect of thermochemotherapy on the two lymphoma variants. However, correlation between degree of malignancy and sensitivity to thermochemotherapy is not a general feature. In contrast to the results obtained in the AKR lymphoma system, in the B16 melanoma the low malignancy variant, F1, was more markedly affected by the combined treatment than the F10 variant. The increased cytotoxic effect of ADR by supranormal temperatures in the F1 variant was shown to be due to an augmented drug uptake. The results suggest that drug resistance in late stages of tumor progression can be overcome by an agent acting on the cell membrane. However, the data also indicate the necessity of assaying cancer treatment modalities, including those designed to circumvent drug resistance, on various tumor system models.

Inhibition of plasma membrane NADH dehydrogenase by adriamycin and related anthracycline antibiotics.

Doxorubicin (adriamycin) is cytotoxic to cells, but the biochemical basis for this effect is unknown, although intercalation with DNA has been proposed This study suggests that the cytotoxicity of this drug may be due to inhibition of the plasma membrane redox system, which is involved in the control of cellular growth. Concentrations between 10(-6) - 10(-7) M adriamycin inhibit plasma membrane redox reactions greater than 50%. AD32, a form of adriamycin which does not intercalate with DNA, but is cytotoxic, also inhibits the plasma membrane redox system. Thus, the cytotoxic effects of adriamycin, which limit its use as a drug, may be based on the inhibition of a transplasma membrane dehydrogenase involved in a plasma membrane redox system.

Increased uptake of vital dye Hoechst 33342 during S phase in synchronized HeLa S3 cells.

Uptake of the vital dye Hoechst 33342 (HO342) was studied in HeLa S3 cells synchronized with hydroxyurea or thymidine. Cell monolayers were incubated with 0.05-0.5 micrograms/ml HO342 for 1-10 min at 37 degrees C, and the fluorescence intensity of DNA-bound intracellular dye was then measured in a flow cytometer. Modal fluorescence intensity normalized to DNA content was used as a measure of the dye uptake. No efflux of the dye from vitally stained cells was observed during 2 hr at 37 degrees C in a monolayer or at 20 degrees C in a suspension. HO342 uptake increased within in 1 hr after the renewal of progression (early S phase), achieved a maximum level in 2 hr (mid-S phase) and returned to the initial level in 4 hr (late S-G2 phase). The pattern of increased uptake of HO342 was similar in cells synchronized with either hydroxyurea or with thymidine. To estimate the relationship between fluorescence and dye uptake, the amount of dye released by Triton X-100 after vital staining was measured. The method for this measurement was based on the simultaneous addition of chicken erythrocytes ("indicator cells") with Triton X-100. Fluorescence intensity of chicken erythrocytes was significantly higher in the mixture containing mid-S phase than in the mixture containing asynchronous or late S-G2 cells. Thus the fluorescence after vital staining with HO342 reflected HO342 uptake and, possibly, fluctuations of membrane permeability. The period of maximal HO342 uptake in synchronized cells was also characterized by a a high sensitivity of cytotoxic effects of adriamycin. Uptake of HO342 at nonsaturating concentrations may be used as a marker of cell sensitivity and resistance to a number of cytotoxic compounds.

DNA repair replication, DNA breaks and sister-chromatid exchange in human cells treated with adriamycin in vitro

The effects of adriamycin (AM) on DNA repair replication, the frequency of sister-chromatid exchange (SCE), the rate of cell proliferation and the frequency of DNA strand breaks were studied in human cells in vitro. No repair replication was observed in lymphocytes exposed to AM in concentrations up to 10 −3 moles/1. DNA repair replication induced by UV and alkylating agents was not affected by a concentration of AM that completely inhibited cell proliferation (10 −6 moles/1). Fibroblasts exposed to AM at 10 −4 moles/1 in the presence of hydroxyurea showed an increase of strand breaks and cross-links in DNA. When AM was added to UV-irradiated fibroblasts, there was an increase of DNA strand breaks in addition to the breaks caused by UV alone. Similar effects were observed in lymphocytes. A dose-dependent increase of SCE was observed in lymphocytes exposed to low concentrations of AM (<10 −7 moles/1). At higher concentrations the increase of SCE levelled off, and cell proliferation became severely inhibited. There was no evidence of removal of SCE-inducing damage in cells exposed to AM during G 0 or G 1. The level of SCE induced in the third cell cycle after treatment with AM was not different from that induced during the first two cell cycles. These results suggest that the various genotoxic and cytotoxic effects of AM are caused by different types of cellular damage. Moreover, AM-induced DNA damage persists for several cell cycles in human cells in vitro and seems to be resistant to repair activity.

Cytotoxic effects of adriamycin on mouse hypoglossal neurons following retrograde axonal transport from the tongue.

We reported recently that the fluorescent, cytostatic drug, adriamycin (Doxorubicin) may reach the hypoglossal neurons by retrograde axonal transport from the nerve terminals of the tongue. The present investigation was undertaken to ascertain whether morphological changes occur in the hypoglossal neurons due to retrograde transport of adriamycin. Neuronal degeneration was observed in the hypoglossal nucleus 14 days after i.m. injection of adriamycin into the tongue. Early neuronal changes, such as rarefaction of the nuclear chromatin and segregation and fragmentation of the nucleolar components, were succeeded by cytoplasmic vacuolation, disappearance of ribosomes and other degenerative features. These observations are important from a neurotoxicologic viewpoint since they demonstrate that retrograde axonal transport may provide a route for the entry of adriamycin into the nervous system. Thus far, adriamycin appears to be the only known substance which can be traced directly in the neurons and cause their degeneration. An experimental method of damaging the motor neurons of the CNS has been introduced. A new toxic model for the investigation of experimental motor neuron disease is therefore available by the use of adriamycin.

Modulation of the cytotoxicity of the antitumor antibiotic peplomycin by membrane-interacting drugs and by increased levels of calcium ions.

The cytotoxic effect of peplomycin (PEP), a new derivative of bleomycin glycopeptide antibiotics, toward HeLa cells and mouse FM3A cells was markedly enhanced by combination of PEP with non-toxic doses of the membrane-interacting drugs verapamil (0.1-0.2mM) and dibucaine (0.15-0.25mM), or with CaCl2 (10-16 mM). Treatment with verapamil or CaCl2 following PEP treatment also effectively enhanced the cytotoxic effect of PEP, suggesting an interaction with PEP-induced damage. Cellular uptake of PEP did not increase in dibucaine-treated cells, suggesting no correlation between membrane permeability to PEP and enhanced cytotoxicity. Increased Ca2+ did not enhance the cytotoxic effects of adriamycin, mitomycin C, cis-diamminedichloroplatinum (II), vinblastine or macromomycin, thus suggesting a unique interaction with PEP. The enhancing action of verapamil and Ca2+ was greatly promoted by 41 degrees hyperthermia, although 41 degrees alone scarcely enhanced PEP cytotoxicity. Cytochalasin B and alpha-tocopherol, but not cytochalasin D, reversed the enhanced cytotoxicity produced by PEP and verapamil or dibucaine, but did not reverse the enhanced cytotoxicity by PEP combined with increased Ca2+. The enhancing action of Ca2+ was, however, antagonized by ruthenium red and lanthanum chloride, which are potent inhibitors of Ca2+ uptake by cells, or by magnesium chloride. Verapamil and increased Ca2+ promoted the decomposition of the DNA-membrane complex induced by PEP in HeLa cells, and also impaired the regeneration of the decomposed DNA complex.