Search

Anthracyclines are among the most useful agents for the treatment of neoplastic disease, but their clinical use is limited by progressive cardiomyopathy. A few studies have suggested the role of microtubules for the understanding of this toxicity. By using kinetic and structural studies, we demonstrate the disorganizing action of fluoro-doxorubicin, a novel anthracycline, on the microtubule system. Microtubules have a rich and complex composition in relation to their numerous functions in cells. In the present study, we investigate the role of two major microtubule-associated protein (MAP) families, Tau and MAP2. Both MAP families are responsible for the properties of different classes of microtubules. We show the differential effect of fluoro-doxorubicin on these two classes of microtubules. Furthermore, we show that fluoro-doxorubicin is able to affect the capacity of purified tubulin to form normal microtubules. This study confirms that anthracyclines may interfer with the microtubule organization. We suggest that some classes of microtubules, with regard to their MAP composition, may be affected more specifically in cardiac myocytes.

Positively charged doxorubicin (DOX) and non-positively charged anthracyclines, aclarubicin (ACR) and morpholino-carminomycin (KRN 8602), have been investigated with respect to pharmacological parameters, cytotoxicity, DNA damage and repair in DOX-sensitive and -resistant murine and human cells. Friend leukemia cells (FLC) resistant to high concentrations of doxorubicin (DOX-RFLC3) or daunorubicin (DNR-RFLC3) (1771 and 1543 fold resistance respectively) express less than 10 fold resistance to aclarubicin (ACR). In these cells, the intracellular accumulation of ACR is similar in sensitive and resistant cells. Resistance to ACR was not observed in either DOX-RFLC1 or DNR1 with a lower level of resistance (27 fold). Increased expression of a 170,000-dalton surface antigen (gp-170) was found to be correlated with the level of resistance. However, when the selective agent in ACR, despite the low level of resistance (2.8 fold) both high expression of gp 170 and resistance to DOX (77 fold) or DNR (62 fold) are observed. It is assumed therefore that induction of multidrug resistance phenotype can be achieved by compounds which do not display cross resistance with DOX or DNR. Reduced levels or absence of cross-resistance can be related to the electrical charge of the compound. This assumption is supported by further studies on DOX-sensitive or -resistant human K562 cells exposed to another non-positively charged anthracycline, KRN 8602. In the continuous presence of drug, K562/DOX were less resistant to KRN 8602 (2.9 fold) than to DOX (31 fold). After short time exposure followed by growth in drug-free medium, absence of cross-resistance to KRN 8602 has been observed in K562/DOX. Furthermore, accumulation experiments showed that high intracellular drug concentrations were rapidly achieved (within 15 min) in both DOX-sensitive and -resistant cells. In cells exposed to DOX, DNA single-strand break (DNA-SSBs) frequencies were related to time and drug concentration while those produced by KRN 8602 or ACR were maximal after short time incubation. DNA-SSBs produced by these anthracyclines are not repaired when cells are incubated in drug free medium. In DOX resistant cells, DNA-SSBs produced by DOX were repaired whereas those produced by ACR or KRN 8602 were not. It is suggested, therefore, that absence of cross resistance to various anthracyclines is related to differences in the chemical electrical charge, which may influence drug accumulation and DNA repair in resistant cells.

In adriamycin-resistant murine (FLC) and human (K562) leukemia cells, phosphatidylcholine increases and phosphatidylethanolamine decreases compared to adriamycin-sensitive lines. This change is due to an increase in phosphatidylethanolamine methylation. The choline pathway of phosphatidylcholine biosynthesis is also disturbed in resistant cells with a blocking step of CDP-phosphocholine transferase and a decrease in sphingomyelinase activity. These changes in phospholipid metabolism are suggested to be responsible for the changes in membrane fluidity reported previously (Tapiero et al, 1986) for resistant cells.

Tumor cell resistance to anthracyclines has been associated with increased activity against free radicals. Here, we have investigated the direct effect of doxorubicin (DOX) in the modulation of glutathione level and antioxidant activities in DOX-sensitive and-resistant cells (288 fold). The glutathione level in untreated cells was 88% greater in resistant than in sensitive cells. The activities of the superoxide dismutase, glutathione -S-transferase and glutathione reductase were respectively 24, 15 and 38% higher in resistant cells than in their sensitive counterparts. In contrast, catalase and total glutathione peroxidase were reduced in resistant cells by 18 and 21% respectively. Moreover, the activity of selenium-dependent glutathione peroxidase was lowered by 47% in the resistant as compared to the sensitive cells. Exposure of sensitive or resistant cells to low doses of DOX did not affect these levels in either cell variant. It is concluded therefore that resistance to anthracyclines may not always be associated with an elevated level of intracellular antioxidant activity enzymes.

The relationship between the intracellular amount of a new anthracycline derivative, 4'-O-tetrahydropyranyl-adriamycin (THP-ADM) and its cytotoxic activity in Friend leukemia cells (FLC) was investigated. By comparison to adriamycin (ADM), the uptake of THP-ADM is a very rapid process reaching maximal levels within 5 min. Both drugs are accumulated and retained in the nuclear fraction. The two main consequences associated to these different uptake rate are: following short-time cell exposure to comparable drug concentration, the higher cytotoxic effect of THP-ADM correlates to the ease with which it crosses the cell membrane; the intracellular amount of THP-ADM but not of ADM decreases with the cell density. These results emphasize the importance of considering drug uptake kinetics and its relationship to cytotoxicity. Studies comparing uptake and efflux of both drugs in ADM-resistant cells showed that THP-ADM extrusion correlate more to cytotoxicity than that of ADM. The relevance of these in vitro findings to clinical application is considered.

Mitochondrial damage was found in Friend leukemia cells treated with rhodamine 123 (Rho 123). In contrast, when cells resistant to the drug were similarly treated, mitochondria were unaffected. These results correlated with higher levels of Rho 123 in sensitive as compared to resistant cells. However, when resistant cells were co-treated with verapamil, intracellular Rho 123 levels reached those of sensitive cells. At these levels mitochondrial damage and subsequent cytotoxicity in resistant cells were the same as in sensitive cells. These data suggest that differences in Rho 123 mitochondrial damage and subsequent cytotoxicity in sensitive and resistant cells result entirely from increased intracellular drug levels and not from differences in mitochondrial sensitivity or other mechanisms.

Based on the fluorescence properties of adriamycin (ADM) and daunorubicin (DNR), uptake in sensitive and resistant Friend leukemia cells (FLC) was studied with the aid of the fluorescence activated cell sorter (FACS II). A quantitative cell by cell fluorescence intensity analysis showed a differential affinity of FLC to ADM and DNR. The cellular uptake of these two drugs was temperature dependent and was not hindered by sodium azide treatment; incorporation into isolated nuclei was not temperature dependent, nor hindered by sodium azide. Friend leukemia cell variants resistant to adriamycin (ADM-RFLC) and to daunorubicin (DNR-RFLC) were developed. The rate of uptake of ADM and DNR across the plasma membrane of these two cell variants was lower than in sensitive cells. Although these cells were crossresistant to both ADM and DNR, the drug-induced fluorescence intensity was distributed differently in the corresponding resistant cell variants. We suggest therefore that resistant is the consequence of changes induced in the plasma membrane components. These changes may differ according to which drug is used.

Using the fluorescent membrane potential probe, 3,3'-dihexyl-oxacarbocyanine (DiOC6(3], we found a 4-fold higher uptake in Adriamycin (ADM)-sensitive versus -resistant Friend leukemia cells (FLC). When sensitive cells were treated in the presence of high potassium (120 mM K+), there was a greater than 80% reduction of DiOC6(3) uptake. Using carbonylcyanide 4-trifluoromethoxy-phenylhydrazone (FCCP), a specific inhibitor of mitochondrial membrane potential, DiOC6(3) accumulation was reduced by less than 30% in these cells. Both results support the conclusion that a greater uptake of DiOC6(3) in ADM-sensitive than in -resistant cells indicates an increased plasma transmembrane potential. Since electronegative plasma membrane potentials are a driving force for the transport of lipophilic positively-charged compounds, differences in membrane potentials between sensitive and multiple drug resistant (MDR) tumor cells could have an important influence on drug accumulation and cytotoxicity. The drugs which our ADM-resistant FLC display multiple drug resistance to are positively charged. In MDR FLC, the calcium channel antagonist, verapamil, has been shown to block the efflux of Rhodamine 123 (Rho 123) and other positively-charged compounds. Since DiOC6(3) is also positively-charged, we used verapamil to investigate its effects on drug uptake. In MDR FLC, verapamil increased DiOC6(3) accumulation by 1.9-fold, whereas in sensitive cells it was increased 1.5-fold. In contrast, verapamil increased the levels of Rho 123 in resistant cells 7.8-fold but lowered them in sensitive cells 1.5-fold. The minimal loss of DiOC6(3) from both sensitive and MDR cells and the above results can best be interpreted as indicating that DiOC6(3) is not transported by the efflux "pump" system but that verapamil induces a plasma membrane potential increase in sensitive and resistant cells that DiOC6(3) is sensitive to. On the other hand, since Rho 123 did appear to be actively effluxed from these resistant cells, the enhancement of this compound by verapamil was more likely due to inhibition of the MDR "pump." How, or whether, plasma membrane potentials and the MDR efflux "pump" are related remains to be investigated. In the resistant cells, verapamil also induced an increase (13-fold) in the accumulation of the electrically neutral fluorescent probe for calcium, INDO-1/AM. However, verapamil had no effect on the efflux of this compound, which was equivalent in both resistant and sensitive cells. Thus, a new effect of verapamil on drug accumulation in MDR cells is identified here.

Variants of Friend leukemia cells (FLC) selected for resistance to either adriamycin (ADM), daunorubicin (DNR) or aclacinomycin A (ACM) by step-wise exposure to each drug, were found to be cross-resistant to ADM and DNR but not to ACM. In addition, an epithelial cell line isolated from normal monkey kidney (CV-1) was found to be intrinsically resistant to ADM and DNR but not to ACM. In contrast, a human breast carcinoma cell line (MCF-7) was found to be sensitive to all three compounds. In these latter cell lines as well as in the FLC variants, lowered intracellular amounts of ADM and DNR correlated with resistance, but ACM levels were the same in sensitive and resistant cells. When cells with either acquired or intrinsic resistance were treated with ACM in combination with ADM or DNR, significant increases in the intracellular amounts of these latter compounds were found. Increased drug accumulation in resistant cells treated this way was accompanied by increased cytotoxicity. When resistant cells were exposed to ACM in combination with other anthracyclines, similar results were obtained. In comparison, these phenomena were not observed when either one of the sensitive cell types (parental FLC and MCF-7) were treated similarly. Since ADM and DNR resistant cells are sensitive to ACM and their resistance circumvented by ACM, this drug may have important clinical applications when used in combination with other anthracyclines.

Friend erythroleukemia cells (FLC) selected by exposure to Adriamycin (doxorubicin) express an approximate 2.5-fold (ARN1) or 13-fold (ARN2) resistance to the drug with various degrees of cross-resistance to other anthracyclines, vinca alkaloids, and epipodophyllotoxins. Because the redox cycling of the quinone moiety of Adriamycin is known to produce oxidative stress, however, an analysis of glutathione (GSH) and related enzyme systems was undertaken in the wild-type and selected resistant cells. In ARN1 and ARN2, superoxide dismutase (SOD) and catalase activities were slightly decreased, intracellular GSH and GSH reductase were essentially unchanged, and total GSH peroxidase, glutathione S-transferase (GST), and DT-diaphorase activities were slightly elevated. In each case there was no stoichiometric relationship between degree of resistance and level of activity. GST isozymes were purified from each cell line by HPLC GSH affinity column chromatography. Two-dimensional gel electrophoresis and western blot immunoreactivity against a battery of GST isozyme polyclonal antibodies determined that both the resistant and sensitive cells expressed isozymes of the alpha, pi, and mu classes (alternative murine nomenclature: M1, M2, M3). Of significance, both ARN1 and ARN2 cell lines expressed a unique alpha subunit which was absent from the parent FLC cell line. This isozyme presumably accounted for the increased GSH peroxidase activity (cumene hydroperoxide as substrate) found in ARN1 and ARN2 and may play a role in the small incremental resistance to melphalan found for both resistant lines. Expression of the isozyme was not stoichiometric with respect to degree of resistance. The presence of this isozyme may contribute to the resistant phenotype or may be the consequence of a more general cellular response to oxidative stress.

Cross-resistance to rhodamine 123 (Rho-123) has been found in Adriamycin (ADM)-resistant and daunorubicin (DNR)-resistant Friend leukemia cell variants. Cytotoxicity in sensitive cells correlates with the intracellular amount of Rho-123, as determined by high-pressure liquid chromatography. Differential resistance coincides with Rho-123 accumulation which in sensitive cells was 20-fold higher than in resistant cells after 180 min of treatment. Sodium azide, which has been shown to inhibit ADM efflux and consequently increase drug accumulation in ADM-resistant cells, did not inhibit Rho-123 efflux. The difference in Rho-123 accumulation between sensitive and resistant cells correlates with cytotoxicity, which is in contrast to what has been found in these cells when treated with either ADM or DNR. Moreover, in contrast to the known effects of ADM and DNR on macromolecular synthesis, Rho-123 in sensitive cells was found to inhibit protein synthesis but had no effect on DNA or RNA synthesis. At Rho-123 doses which inhibited protein synthesis, drug localization changed from mitochondrial specific to generalized cytoplasmic. This effect was never achieved in resistant cells, even with prolonged drug exposure. The relevance of these findings is that different mechanisms of resistance to different drug types can be identified in the same cells even though similar resistance occurs. The similarity in resistance need not share a common mechanism. Although the drugs are effluxed more efficiently in resistant cells, the mechanisms for resistance in each case seem to differ. In the case of ADM and DNR, it appears to be multifactorial, whereas with Rho-123, total intracellular accumulation seems to be most important.

Intracellular accumulation of adriamycin (ADM) was found to be increased in human breast carcinoma cell line (MCF-7) and cardiac-muscle cells as compared to an epithelial cell line derived from normal monkey kidney (CV-1) and non-muscle cells (fibroblasts) derived from the heart. This increase correlates with greater sensitivity of the carcinoma cell line to ADM. In CV-1, efflux of ADM contributes to low drug accumulation which correlates with the intrinsic drug resistance of the cells. Blockage of ADM efflux in this resistant cell line and subsequent increases in intracellular accumulation and sensitivity can be achieved by co-treatment with verapamil. ADM accumulation and sensitivity in MCF-7 however cannot be significantly increased by verapamil. These data demonstrate selectively for ADM in human breast carcinoma cell line, MCF-7 and cardiac-muscle cells in vitro. The reversal by verapamil of the normal cells' natural resistance, may have importance in the clinical use of verapamil as a resistance modulating agent.

Chromosome analysis was performed on Friend leukemia cells sensitive and resistant to doxorubicin. With increasing levels of resistance, increased numbers, of metacentric chromosomes and several chromosomal markers were observed. When sensitive cells were exposed to a toxic dose of doxorubicin, multiple chromosomal breaks were observed in 62% of cells. In contrast, when doxorubicin resistant cells were exposed to cytotoxic concentrations, the pulverization phenomenon was not observed. This striking difference suggests a different mechanism for cytotoxicity in sensitive and resistant cells. Moreover, when logarithmically growing cells were grafted subcutaneously in DBA2 mice, the tumorigenic property was related to the level of doxorubicin resistance.

Friend leukemia cells (FLC) resistant to different concentrations of doxorubicin were used to investigate the biochemical and biophysical changes associated with resistance. We have found that fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene analyzed on single cell level increased in resistant as compared to sensitive FLC. Furthermore, phospholipid analysis of sensitive and doxorubicin-resistant cells revealed changes in ratios of phosphatidyl-choline to phosphatidyl-ethanolamine and phosphatidyl-choline to sphingomyelin. These results correlate with decreased electrophoretic mobility in resistant cells. Our results indicate that changes in cell structure occur with the level of resistance to doxorubicin. These changes are probably the consequence rather than the cause of resistance.

Pleiotropic resistance to rhodamine 123 (Rho-123) in Adriamycin (ADM)-resistant Friend leukemia cells was circumvented by cotreatment with 10 microM verapamil. Increased cytotoxicity corresponded to higher intracellular Rho-123 levels. The verapamil-induced increase of drug accumulation in resistant cells is accounted for at least in part by the blockage or slowing of Rho-123 efflux from these cells. In contrast, accumulation and consequent cytotoxicity of Rho-123 in sensitive cells are not increased by verapamil. Similar results were obtained when ADM was used in this cell system. These results suggest that the efflux system for Rho-123 and ADM in sensitive cells is either reduced or absent. Although Rho-123 accumulates specifically in mitochondria and ADM mainly in the nucleus, the loss of these two different classes of compounds from resistant cells appears to occur via a similar or common mechanism. The similarities in drug transport between Rho-123 and ADM may have important implications when applied to an in vivo environment.

THP-ADM is a new anthracycline with broad antitumor activity without cardiac toxicity or alopecia in experimental models. Phase I studies had established a proposed dose for phase II trials of 50 mg/m2 every three weeks. This modality gave an insignificant result in breast carcinoma. Cellular pharmacokinetics suggested that a longer time of administration could be more efficient. In this phase II trial oriented to advanced breast cancer, we have used 3 consecutive daily doses of 20 mg/m2/day in monthly cycles with dose escalation in each patient. We have observed 28% partial remissions (PR). Two patients previously treated with adriamycin had PR. Significantly less alopecia and no cardiac toxicity were observed.

The purpose of these studies was to investigate whether the cell-differentiating effect of anthracyclines can trigger an over-production and secretion of molecules that may interfere with the tumor-host relationship. We exposed mouse hybridoma B-cells, which are devoted to immunoglobulin production, to doxorubicin (10-40 ng/ml). We found that most doxorubicin-treated cells secreted 3- to 5-fold higher amounts of immunoglobulin than untreated cells, along with an accumulation of 50% of them in the G2+M phase of the cell cycle. The antigenic specificity of the immunoglobulin and its size pattern as determined by polyacrylamide gel electrophoresis were similar whether or not cells were treated with doxorubicin. The enhancement of immunoglobulin secretion by doxorubicin was associated with an increase of the intracellular pool of heavy and light chains of the immunoglobulin. Furthermore, an elevated synthesis of immunoglobulin was observed. The synthesis of other proteins also appeared to be modified in these circumstances. These data suggest that doxorubicin can potentiate the biological functions of target cells when used at low concentrations, elevating the production and secretion of effector molecules that interfere with the tumor-host relationship.