Sensitization to the cytotoxicity of melphalan by ethacrynic acid and hyperthermia in drug-sensitive and multidrug-resistant Chinese hamster ovary cells.

The ability of physical and pharmacological modulators to increase the cytotoxicity of melphalan was investigated in Chinese hamster ovary cells using a clonogenic cell survival assay. Hyperthermia has potential for use in cancer treatment, particularly as an adjuvant to chemotherapy or radiotherapy. Ethacrynic acid is a glutathione S-transferase inhibitor and also undergoes conjugation with glutathione. Interactions between hyperthermia (41-43 degrees C), ethacrynic acid and melphalan were evaluated in multidrug-resistant (CH(R)C5) cells with overexpression of P-glycoprotein (33.69-fold), and in drug-sensitive (AuxB1) cells. GST alpha was expressed at a higher level (3.65-fold) in CH(R)C5 cells than in sensitive cells, whereas levels of isoforms pi and mu were the same. GST pi was the most highly expressed isoform in the two cell populations. Ethacrynic acid was cytotoxic at elevated temperatures, while it caused little or no cytotoxicity at 37 degrees C. This effect occurred in drug-resistant and drug-sensitive cells, and attributes thermosensitizing properties to ethacrynic acid. Ethacrynic acid (20 microM) alone did not alter the cytotoxicity of melphalan at 37 degrees C. Hyperthermia potentiated drug cytotoxicity in cells, both with and without ethacrynic acid treatment. Ethacrynic acid could be useful in cancer treatment by acting as a thermosensitizer when combined with heat and by enhancing the cytotoxicity of melphalan at elevated temperatures. A major advantage arising from the use of regional hyperthermia is the ability to target drug cytotoxicity to the tumor volume. A useful finding is that ethacrynic acid, heat and/or melphalan are also effective against multidrug-resistant cells with overexpression of P-glycoprotein.

Immobilization of native and poly(ethylene glycol)-treated ('PEGylated') bovine serum amine oxidase into a biocompatible hydrogel.

Native bovine serum amine oxidase (BSAO) and poly(ethylene glycol) (PEG)-treated ('PEGylated') BSAO were immobilized into a hydrogel during its synthesis. The hydrogel was obtained by cross-linking of BSA with PEG di-nitrophenyl carbonates with a molecular mass of 10 kDa. Approx. 60% of the amino groups at the surface of BSAO were modified by monomethoxy-PEG with a molecular mass of 5 kDa when the reaction was carried out for 5 h in borate buffer, pH 9. The number of anchorage points of BSAO in the matrix, which was determined as minimal when PEGylated BSAO was used or maximal when native BSAO was used, did not influence the apparent K(m) and V(max) values of the different preparations. The apparent K(m) values of both forms of the enzyme were decreased due to preconcentration of benzylamine substrate by the negatively charged hydrogel. V(max) values were generally lower upon immobilization. We can therefore conclude that the hydrogel swelling has no significant effect on the enzyme's structure. The operational stability, evaluated in the presence of substrate, was generally increased upon enzyme immobilization into the hydrogel. Enzymic hydrogels were very stable during storage in solution at 4 degrees C, maintaining a high activity even after several weeks. The immobilization of both forms of BSAO did not improve their thermostability at 65 degrees C. The BSA-PEG hydrogel is a good matrix for immobilization of enzymes with therapeutic potential such as BSAO.

Amine oxidase, spermine, and hyperthermia induce cytotoxicity in P-glycoprotein overexpressing multidrug resistant Chinese hamster ovary cells.

Multidrug resistance is a major obstacle for the successful use of chemotherapy. The multidrug resistance phenotype is often attributed to overexpression of P-glycoprotein, which is an energy-dependent drug efflux pump. We investigated a new strategy to overcome multidrug resistance, using purified bovine serum amine oxidase, which generates two major toxic products from the polyamine spermine. The cytotoxicity of the aldehyde(s) and H2O2, produced by the enzymatic oxidation of micromolar concentrations of spermine, was evaluated in multidrug resistant Chinese hamster ovary cells CHRC5 with overexpression of P-glycoprotein, using a clonogenic cell survival assay. We examined the ability of hyperthermia (42 degrees C), and inhibition of cellular detoxification systems, to sensitize multidrug resistant cells to spermine oxidation products. Severe depletion of intracellular glutathione was achieved using L-buthionine sulfoximine and inhibition of glutathione S-transferase by ethacrynic acid. CH(R)C5 cells showed no resistance to the toxic oxidation products of spermine, relative to drug-sensitive AuxB1 cells. Exogenous catalase protected cells against cytotoxicity of H2O2, but spermine-derived aldehyde(s) still caused some cytotoxicity. Hyperthermia (42 degrees C) enhanced cytotoxicity of spermine oxidation products. Cytotoxic responses in CH(R)C5 cells were compared to the drug-sensitive cells, to determine whether there are differential responses. CH(R)C5 cells were more sensitive to the cytotoxic effect of spermine oxidation products under more extreme conditions (higher temperature, higher spermine concentration, and longer exposure time). Glutathione depletion or glutathione S-transferase inhibition also led to enhanced cytotoxicity of spermine oxidation products in CH(R)C5 and AuxB1 cells. Our findings suggest that hyperthermia, combined with toxic oxidation products generated from spermine and amine oxidase, could be useful for eliminating drug-sensitive and multidrug resistant cells.

Correlation between glutathione and stimulation of the pentose phosphate cycle in situ in Chinese hamster ovary cells exposed to hydrogen peroxide.

The effect of glutathione on stimulation of pentose phosphate cycle activity during oxidative challenge was evaluated in intact Chinese hamster ovary cells in situ. Glutathione was depleted to varying levels with L-buthionine-[S,R] sulfoximine. The level of stimulation of pentose phosphate cycle activity by exogenous H2O2 (4 mumol/10(7) cells) was dependent on the time of pretreatment with L-buthionine-[S,R] sulfoximine and was proportional to the total glutathione concentration. This was not related to the amount of GSSG, since its level was exceedingly low under conditions where H2O2 stimulated pentose phosphate cycle activity. The amount of GSSG in cells increased after exposure to 10-fold higher concentrations of H2O2 under conditions where there was no stimulation of pentose phosphate cycle activity above the basal level. Paraquat caused stimulation of pentose phosphate cycle activity which was independent of L-buthionine-[S,R] sulfoximine pretreatment and of the glutathione content of cells. The stimulatory effects of both oxidants on pentose phosphate cycle activity appeared to be independent of glutathione reductase activity since they were unaffected in cells treated with 1,3-bis(2-chloroethyl)-1-nitrosourea. The inhibitory effect of L-buthionine-[S,R] sulfoximine on stimulation of pentose phosphate cycle activity by H2O2 did not appear to be due to the inhibitor itself, but rather to the overall level of glutathione. Glutathione could have a role in maintaining activity of the pentose phosphate cycle at a level which is appropriate for the severity of the oxidative challenge as well as for the capacity of the cellular antioxidant defenses.

Glucose, glutathione, and cellular response to spermine oxidation products.

Bovine serum amineoxidase (BSAO) oxidatively deaminates polyamines, which contain primary amine groups with formation of several toxic products, H2O2, and aldehyde(s). We evaluated the role of glucose metabolism via the pentose phosphate cycle and the level of intracellular glutathione on cytotoxicity induced by each of the toxic products in Chinese hamster ovary (CHO) cells. Glucose protected cells against cytotoxicity in the presence of BSAO at low spermine concentrations ( < 50 microM), where H2O2 was the only toxic species present. When catalase was present, cytotoxicity is attributed to spermine-derived aldehyde(s). Glucose did not protect cells against cytotoxicity induced by spermine-derived aldehyde(s), nor by the aldehyde acrolein. Hydrogen peroxide produced by spermine and BSAO stimulated pentose cycle activity, whereas the aldehyde(s) did not. Depletion of intracellular glutathione with L-buthionine sulfoximine (1 mM, 24 h) sensitized cells to the cytotoxic effects of both H2O2 and the aldehyde(s) produced by spermine and BSAO. The pentose cycle and the glutathione redox cycle have an important role in protection against H2O2 generated from spermine oxidation. Glutathione appears to have a role in protecting cells against cytotoxicity attributed to spermine-derived aldehyde(s), most likely by conjugation in a reaction catalyzed by glutathione S-transferase, whereas metabolism of glucose via the pentose cycle did not. The metabolism of both glucose and glutathione, affect the cellular response to H2O2 and aldehyde(s) derived from spermine, although different pathways are involved.

The effect of hyperthermia and verapamil on melphalan cytotoxicity and transport in multidrug-resistant Chinese hamster ovary cells.

The effect of both hyperthermia and verapamil on cytotoxicity and transport of melphalan was studied in a pleiotropic drug-resistant Chinese hamster ovary cell line (CHRC5) and in the drug-sensitive parent line (AuxB1). The CHRC5 cell line was selected for resistance to colchicine but is also cross-resistant to other drugs including melphalan. Verapamil (10 microM) increased melphalan cytotoxicity in drug-resistant cells but not in drug-sensitive cells. Hyperthermia (40 to 45 degrees C) increased melphalan cytotoxicity in both cell lines. In drug-resistant but not drug-sensitive cells, melphalan cytotoxicity was increased further when verapamil was combined with hyperthermia (40 to 45 degrees C). The increased cytotoxicity caused by verapamil in drug-resistant cells was accompanied by alterations in membrane permeability to melphalan. The cellular uptake of melphalan after 15 min increased in the presence of verapamil (7 to 30 microM) at 37 and 42 degrees C. When verapamil (10 microM) was present, the rate of efflux of melphalan from CHRC5 cells decreased by almost 40% at 37 degrees C. The rate of efflux was increased at 42 degrees C relative to 37 degrees C, but with verapamil the rate decreased to that obtained at 37 degrees C in CHRC5 cells. In drug-sensitive cells, verapamil (< or = 50 microM) did not affect either uptake or efflux of melphalan. These findings suggest that verapamil could be beneficial by increasing the effectiveness of melphalan in the elimination of multidrug-resistant cells. The combination of hyperthermia and verapamil could be especially advantageous by increasing melphalan cytotoxicity in a localized target region.

P-glycoprotein of blood brain barrier: cross-reactivity of Mab C219 with a 190 kDa protein in bovine and rat isolated brain capillaries.

P-glycoprotein (P-gp), an active efflux pump of antitumor drugs, is strongly expressed in endothelial cells of the blood brain barrier (BBB). Two proteins (155 and 190 kDa) were detected by Western blot analysis of beef and rat capillaries with the monoclonal antibody (MAb) C219. In order to characterize the nature of these proteins, their profile of solubilization by different detergents was established and compared with that of P-gp from the CHRC5 tumoral cell line. The 155 kDa protein (p155) of capillaries and the P-gp of CHRC5 cells were well solubilized by deoxycholate and Elugent, whereas the 190 kDa kDa protein (p190) was only solubilized by sodium dodecylsulfate (SDS). Both proteins have different patterns of extraction by Triton X-114, p155 partitioning as a membrane protein, while p190 was insoluble. Deglycosylation of capillary proteins resulted in a 27-28 kDa decrease in the apparent molecular weight of p155, similar to that observed for the P-gp of CHRC5 cells, but a decrease of only 7-8 for p190. Only p155 was immunoprecipitated by MAb C219. These results suggest that only p155 is the P-gp in BBB and that MAb C219 cross-reacts with a 190 kDa MDR-unrelated glycosylated protein. Consequently, the use of this antibody, which is frequently used to detect P-gp in tumors, could be a pitfall of immunohistochemistry screening for cancer tissues and lead to false positive in the diagnosis of MDR.

Heat enhancement of cytotoxicity induced by oxidation products of spermine in Chinese hamster ovary cells.

This study investigates the potential of using polyamines as thermosensitizers, in the presence of bovine serum amine oxidase (BSAO), as a new anticancer strategy. The effect of hyperthermia on cytotoxicity of spermine oxidized by purified bovine serum amine oxidase was investigated in Chinese hamster ovary cells. Several different spermine concentrations were employed in the presence of BSAO at 37 degrees and 42 degrees. Cytotoxicity was considerably enhanced at 42 degrees. Heat also increased the individual cytotoxicity of both exogenous H2O2 and the exogenous aldehyde acrolein. Thus, both of these species could contribute to the thermal enhancement of cytotoxicity caused by BSAO and spermine. The effect of temperature was especially marked in the presence of exogenous catalase. This cytotoxicity cannot be accounted for by H2O2 and was attributed to aldehyde(s). The involvement of aldehyde(s) in cytotoxicity at 42 degrees was also confirmed by the complete inhibition of cytotoxicity with both exogenous aldehyde dehydrogenase and exogenous catalase. A particularly interesting finding, in the presence of exogenous catalase, was that conditions of BSAO and spermine (< or = 50 microM) which were non-toxic at 37 degrees became cytotoxic at 42 degrees. This suggests that spermine-derived aldehyde(s), that were non-toxic at 37 degrees, contributed to cytotoxicity at 42 degrees and resemble thermosensitizers. The thermosensitizing activity of aldehyde(s) produced in the BSAO-catalysed oxidation of spermine has potential value for improving the therapeutic effects of hyperthermia and could be considered for future application in cancer therapy. Polyamines are present at elevated levels in tumour cells and have been considered as heat sensitizers. By delivering BSAO into tumour cells, toxic oxidation products of polyamines could be produced in situ for selective killing of tumour cells.

The role of glucose in cellular defences against cytotoxicity of hydrogen peroxide in Chinese hamster ovary cells.

The presence of glucose in the cellular environment influences the response of cells to hydrogen peroxide. This study examines the effect of glucose on clonogenic cell survival of Chinese hamster ovary cells exposed to micromolar concentrations of exogenous hydrogen peroxide. Exposure to hydrogen peroxide (20 mumol/10(7) cells) resulted in considerable cytotoxicity that was unaffected by the presence or absence of glucose. However, glucose protected the cells from killing induced by milder exposure (1 mumol/10(7) cells) to the oxidant causing a shift in the dose-response curve. This effect was considered in terms of glucose metabolism via the pentose phosphate cycle. A low nontoxic concentration of hydrogen peroxide (0.1 mumol/10(7) cells) markedly increased pentose phosphate cycle activity in normal cells. Clonogenic survival and activity of the cycle for cells depleted of total glutathione to about 8.6% of its initial value and/or with catalase activity reduced to about 10% of control levels were also determined. Neither of these modifications alone completely abolished the protective effect of glucose. Efficacy of glucose protection against cytotoxicity of hydrogen peroxide diminished in cells depleted of glutathione, and this was not accompanied by any detectable increase in pentose phosphate cycle activity above the control level. Cells depleted of catalase alone had a profile of survival and pentose phosphate cycle activity similar to that of control cells when exposed to hydrogen peroxide. Cells depleted of both glutathione and catalase were almost as sensitive to hydrogen peroxide as the cells incubated without glucose. They also did not express any detectable increase in pentose phosphate cycle activity. Survival of those cells, when exposed to hydrogen peroxide, was almost the same regardless if glucose was present or not. These results demonstrate an important role for the glutathione redox cycle, catalase, and the pentose phosphate cycle in protection against hydrogen peroxide in Chinese hamster ovary cells. They confirm the essential role of glucose and pentose phosphate cycle activity for the detoxification of hydrogen peroxide via the glutathione redox cycle. The data suggest that the ability of catalase to metabolise peroxide may also depend on metabolism of glucose via the pentose phosphate cycle. A clear understanding of the protective mechanisms in cells against hydrogen peroxide has many applications since this common reactive oxygen species is implicated in several pathophysiologies and in the action of certain chemotherapeutic drugs.

Aldehyde dehydrogenase and cytotoxicity of purified bovine serum amine oxidase and spermine in Chinese hamster ovary cells.

Bovine serum amine oxidase (EC 1.4.3.6) catalyses the oxidative deamination of polyamines giving rise to the corresponding aldehydes, ammonia, and hydrogen peroxide. It has been suggested that the dialdehyde produced during the oxidation of spermine subsequently undergoes spontaneous beta-elimination to form acrolein. Oxidation of the aldehydes by aldehyde dehydrogenase (EC 1.2.1.5) thus eliminates these reactive species and prevents the formation of acrolein. This work studies the role of each of the oxidation products of spermine in cytotoxicity induced by purified bovine serum amine oxidase. The inhibition patterns of NAD-dependent aldehyde dehydrogenase and catalase against cytotoxicity of bovine serum amine oxidase were determined in Chinese hamster ovary cells at 37 degrees C. Cytotoxicity caused by exogenous hydrogen peroxide, added directly (> 10 microM) or generated by glucose oxidase (0.5 U/mL), was completely inhibited by catalase. Cytotoxicity caused by bovine serum amine oxidase (5.7 x 10(-3) U/mL) and spermine (340 microM) was completely inhibited by catalase only during short incubation times after which time cytotoxicity occurred. This indicates that hydrogen peroxide was the only species contributing to cytotoxicity at this stage of the reaction. Aldehyde dehydrogenase alone caused partial inhibition of cytotoxicity, but only later in the reaction. Cytotoxicity was completely eliminated in the presence of both catalase and aldehyde dehydrogenase. Exogenous acrolein (> 50 microM) also caused cytotoxicity in Chinese hamster ovary cells. However, hydrogen peroxide was toxic to cells at lower concentrations and at shorter exposure times relative to aldehydes. These data show that both peroxide and aldehydes contribute to cytotoxicity of oxidation products of spermine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxicity and kinetic analysis of purified bovine serum amine oxidase in the presence of spermine in Chinese hamster ovary cells.

Bovine serum amine oxidase (BSAO, EC 1.4.3.6) catalyzes the oxidative deamination of polyamines giving rise to the corresponding aldehydes, ammonia and hydrogen peroxide (H2O2). This study demonstrates that amine oxidase (BSAO) purified from bovine serum and exogenous spermine caused cytotoxicity in Chinese hamster ovary (CHO) cells. Cytotoxicity occurred when cells were exposed to BSAO (0.0164-16.4 micrograms/ml) in the presence of spermine (1.9-340 microM). BSAO and spermine alone were not toxic at these concentrations. Cytotoxicity was dependent on the concentration of spermine and on the incubation time, and was also accelerated at 42 degrees C relative to 37 degrees C. Kinetic analysis of the enzymatic reaction, as a function of spermine concentration, showed Michaelis-Menten saturation kinetics. The apparent Vmax increased from 19.1 +/- 0.4 microM min-1 at 37 degrees C to 23.0 +/- 0.3 microM min-1 at 42 degrees C. The apparent Km decreased from 25.5 +/- 2.6 microM at 37 degrees C to 17.7 +/- 1.3 microM at 42 degrees C. Catalase inhibited cytotoxicity, suggesting that H2O2 was partially responsible for cytotoxicity. This work shows that the oxidation products of polyamines, rather than the polyamines themselves, are responsible for cytotoxicity in mammalian cells. The significance of this study is that amine oxidases could have therapeutic potential in cancer treatment regimens and a beneficial effect is likely when the enzyme is used together with clinical hyperthermia.