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Circumvention of multidrug resistance and reduction of cardiotoxicity of doxorubicin in vivo by coupling it with low density lipoprotein
Life Sciences 72 (2002) 677 – 687 www.elsevier.com/locate/lifescie
Circumvention of multidrug resistance and reduction of cardiotoxicity of doxorubicin in vivo by coupling it $ with low density lipoprotein Elka H.K. Lo a, Vincent E.L. Ooi b, K.P. Fung a,* a
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China b Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong, China Received 12 December 2001; accepted 24 July 2002
Abstract Doxorubicin (Dox) was coupled into human low density lipoprotein (LDL) to form a complex LDL-Dox. In in vitro studies, the accumulation of LDL-Dox in human resistant hepatoma (R-HepG2) cells was found to be higher than that of free Dox in the cells, resulting in an increase of the cytotoxic effect on the cells. Moreover, in in vivo studies, under the same dosage of drugs (1 mg/kg), the anti-proliferative effect on the tumor cells of LDL-Dox in nude mice bearing R-HepG2 cells was higher than that of free Dox as evidenced by the larger reduction in tumor volumes and tumor weights in LDL-Dox treated group. Histological studies showed that LDL-Dox treatment did not cause any heart damage when compared with the control group. In contrast, Dox treatment caused disruption and vacuolization of myocardial filament. Plasma lactate dehydrogenase activity and plasma creatine kinase activity in nude mice bearing R-HepG2 cells were found to be elevated in the Dox-treated group but remained unchanged in LDL-Dox-treated group. The present studies indicate that when Dox is coupled with LDL, the multidrug resistance can be circumvented and the cardiotoxicity can be reduced. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Doxorubicin; Low density lipoprotein; Human resistant hepatoma cells
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This work was supported by an earmarked grant from Research Grants Council (CUHK4060/97M, Hong Kong, China). * Corresponding author. Tel.: +852-2603-6873; fax: +852-2603-5123. E-mail address: [email protected] (K.P. Fung).
0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 1 8 0 - X
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Introduction Antitumoral drugs always exhibit adverse side effects on normal cells because of the lack of selectivity. Moreover, the drug resistance is usually a problem in chemotherapy. It has been an urgent need to develop a target carrier for antitumoral drugs in order to increase the specificity of drugs and the chance of circumvention of multidrug resistance on tumor cells and to decrease the drugs’ toxicities to normal cells. Low density lipoprotein (LDL) was proposed as a target carrier to deliver antitumoral drugs because many studies showed that the cancer cells have a higher number of low density lipoprotein receptors (LDL-R) than the normal cells [1–4]. Doxorubicin (Dox) is one of the prototypes of anthracycline anticancer antibiotic with anti-tumoral activity which is isolated from strains of Streptomyces peucetius [5]. Dox is currently used widely in treating different tumors. The multiple potential mechanistic actions of Dox include the inhibition of activities of DNA topoisomerase I [6] and II [7], inhibition of helicases [8], alteration of membrane structure and function [9] and generation of free radicals [10]. Since the hearts contain high content of oxygen, a large amount of free radicals are produced by Dox causing cadiotoxicity which is the main adverse side effect of Dox, especially during prolonged treatment in the patients. Development of resistance to the multiple antitumoral drugs in cancer cells after prolonged treatment is another major problem of chemotherapy. One of the reasons of multidrug resistance is the overexpression of P-glycoprotein (P-gp) [11]. P-gp can exert active drug efflux out of resistant cells in order to reduce the accumulation of various types of antitumoral drugs and thus preventing them from reaching their cellular targets [12]. In previous studies, we found that LDL could serve as a target carrier for Dox to kill tumor cells in nude mice bearing human hepatoma HepG2 cells [13]. In the present study, we further investigate the cytotoxicity of LDL-Dox on R-HepG2 cells and to examine whether LDL-Dox can circumvent the drug resistance in this resistant cell line. Moreover, we have made use of the R-HepG2 tumor cells bearing nude mice model to investigate the anti-proliferative effect of LDL-Dox on tumor cells and its cardiotoxicity when compared with free Dox.
Methods Cell culture The human resistant hepatoma cell line (R-HepG2) was developed in our laboratory by incubating HepG2 cells in stepwise increasing concentrations of doxorubicin [14]. Treatments were repeated until the cell grew at 1.2 AM doxorubicin. R-HepG2 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1.2 AM doxorubicin. The resulting resistant cells were found to express high content of P-gp by Northern and Western analysis, and exhibit resistance to Dox [14]. Formation of LDL-Dox complex The method reported in Refs. [15,16] was adopted with minor modifications [13]. Ten milligram of doxorubicin was dissolved in 1 ml of autoclaved double distilled water. LDL was mixed with Dox at the
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ratio of 10:1 (w/w). The mixture was incubated in shaking air-bath at 37 jC for 24 hours in dark. The mixture was then loaded onto gel filtration column with G25 Sephadex to separate the free Dox from the LDL-Dox complex. Fractions of 0.5 ml were collected. The LDL-Dox complex so obtained was sterilized by passage through a 0.45 Am acetate millipore filter. After Dox was coupled into LDL, its concentration in the complex was measured. Twenty microlitre of LDL-Dox complex was added to 780 Al of acidified isopropanol. A standard curve of the concentration of doxorubicin in acidified isopropanol versus the absorbance (O.D.) at wavelength 480 nm was obtained. From the curve, the concentration of LDL-Dox was determined. Determination of the accumulation level of Dox in cultured R-HepG2 cells after Dox and LDL-Dox treatments by flow cytometry and confocal laser scanning microscope R-HepG2 cells were seeded in a 6-well plate and incubated at 37 jC, 5% CO2 overnight. The cells were washed twice with PBS. The medium was replaced by RPMI 1640 medium with 10% lipoprotein deficient fetal bovine serum (LPDS) and incubated for further 48 hours at 37 jC, 5% CO2. After that, the medium was removed and the cells were washed twice with PBS. Then, 2 ml of 1 AM of LDL-Dox or Dox in RPMI with 10% LPDS were added and incubated for 1 hour at 37 jC. The adhered cells were trypsinised and the fluorescence intensities of Dox in the cells were analyzed by flow cytometry with FAC Sort (BECTON DICKINSON). Forward and side scatters were used to establish size gates and exclude debris from the analysis. The excitation wavelength was 488 nm while the emitted fluorescence was collected at 585 nm. For confocal microscopic analysis, R-HepG2 cells were first seeded on cover glasses. After the same treatment protocol, cell images were acquired on Multiprobe 2001 from Molecular Dynamics. An excitation filter with 488 nm wavelength and a long-pass emission filter of 510 nm were used. After scanning, images were processed by an image analysis software [13]. Determination of survival of cultured R-HepG2 cells after Dox and LDL-Dox treatments The survival of R-HepG2 cells was determined by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay as described before [13]. The results were presented as mean of percentage of cell survival F S.D. The percentage of cell survival was calculated by dividing the absorbance of treated cells and the absorbance of untreated cell multiplied by 100. Effects of Dox and LDL-Dox on tumor growth in nude mice bearing R-HepG2 cells Nu/nu nude mice (with body weight of 20–25 g and aged 6–8 weeks) were kept in autoclaved cage with polyester fiber filters to avoid contact with the pathogens. All the animal diet (PICO LAB@ Rodent Diet) and tape water were autoclaved before feeding to nude mice ad libitum. Suspension of 8 106 of human resistant hepatoma R-HepG2 cells were injected subcutaneously (s.c.) into the anterior part of the shoulder of the nude mice. Drug treatments were started 2 days after implantation of cells. The mice were then randomly divided into treatment group and control group with 5 mice in each group. Free Dox at dosage 1 mg/kg and 2 mg/kg as well as LDL-Dox at dosage 1 mg/kg in saline were injected into the mice intravenously on 1 injection per 2 days schedule. The injections were given for 4 weeks. Tumor volume was measured by Vernier caliper weekly and tumor volumes
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were calculated according to the formula [17]: Tumor volume (mm3) = a/2 b/2 h k, where a, b and h are the minor and major dimensions and height of the tumor, respectively, k = 3.1416. After the treatment, the nude mice were anesthetized with diethyl ether and the weights of the tumors were recorded. Investigation of the myocardial injury in nude mice after Dox and LDL-Dox treatments Myocardial injury was assessed by measuring the leakage of lactate dehydrogenase (LDH) and creatine kinase (CK) from the heart to the blood. After treatment for four weeks, the tumor-bearing nude mice were anesthetized and the blood was collected in heparinized tube. Plasma LDH and CK activity were then assayed by using LDH assay kit and CK assay kit (SIGMA), respectively. Histological analysis of the hearts in nude mice after Dox and LDL-Dox treatments Another group of tumor-bearing nude mice were sacrificed by cervical dislocation on the next day after the last injection of drug. Hearts were removed immediately and the sections were prepared and stained by standard histological method [18]. The sections were observed under light microscope (OLYMPUS) by different combinations of magnification values of the eye pieces and objective lens of the microscope. Micrographs of the heart sections under microscope were taken for record and comparison [18]. Statistics Student’s t test was used to compare the data obtained in Dox-treated and LDL-Dox-treated groups.
Results The comparison of the accumulation of Dox and LDL-Dox in R-HepG2 cells The accumulation of Dox and LDL-Dox in R-HepG2 cells were determined by flow cytometry. As shown in Fig. 1, there was no significant difference in fluorescent intensity of R-HepG2 cells when incubated with or without Dox. In contrast, an increase in fluorescent intensity was observed in cells treated with LDL-Dox than that treated with free Dox. Fig. 2 shows the result of confocal laser scanning microscopic images. It was found that R-HepG2 cells treated with LDL-Dox exhibited a higher degree of fluorescent intensity than that with free Dox. Moreover, the accumulated free Dox was found to be located mainly near the cell membrane. This result suggests that the intracellular accumulation of LDL-Dox was higher than that of free Dox in R-HepG2 cells. Comparison of cytotoxicity of Dox and LDL-Dox on R-HepG2 cells The percentage cell survival after Dox or LDL-Dox treatment was determined by MTT assay (Figs. 3 and 4). For 24 hours incubation, the IC50 of Dox and LDL-Dox were found to be 368 AM and 48
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Fig. 1. The quantitative analysis of Dox and LDL-Dox accumulated in R-HepG2 cells. The accumulation of Dox and LDL-Dox after 1 hour incubation were measured by flow cytometry.
AM, respectively. When the incubation time was prolonged from 24 hours to 48 hours, the IC50 of Dox and LDL-Dox were found to be 300 AM and 15 AM, respectively. After 72 hours incubation, IC50 of Dox and LDL-Dox were found to be 240 AM and 7 AM, respectively. These results showed
Fig. 2. The confocal laser scanning microscopic analysis on the accumulated level of Dox and LDL-Dox in R-HepG2 cells after 1 hour incubation. Upper panel: Dox-treated R-HepG2 cells; Lower panel: LDL-Dox-treated R-HepG2 cells.
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Fig. 3. The cytotoxicity of Dox on R-HepG2 cells after 24, 48 and 72 hours incubation. The percentage of cell survival was analyzed by MTT assay. x—x, 24 hours; n—n, 48 hours; E—E, 72 hours. The result is presented as mean F S.D. (n = 5).
that as the concentration of Dox and LDL-Dox increased, the numbers of cell viability were decreased, i.e. the percentage of cell survival was dose- and time-dependent for both free Dox and LDL-Dox treatments. In addition, the IC50 of LDL-Dox was lower than that of free Dox at different time points. This result indicated that after Dox was coupled into LDL, its cytotoxic effect in RHepG2 cells could be enhanced i.e. the multidrug resistance of R-HepG2 cells could be circumvented.
Fig. 4. The cytotoxicity of LDL-Dox on R-HepG2 cells after 24, 48 and 72 hours incubation. The percentage of cell survival was analyzed by MTT assay. x—x, 24 hours; n—n, 48 hours; E—E, 72 hours. The result is presented as mean F S.D. (n = 5).
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Treatment of tumor-bearing nude mice with free Dox and LDL-Dox The anti-proliferative effect of Dox and LDL-Dox on the growth of R-HepG2 cells in tumor bearing nude mice was investigated by measuring the tumor volume and tumor weight in the mice after treatment for 4 weeks. Fig. 5 and Fig. 6 showed that there was no significant difference in tumor volume and tumor weight between 1 mg/kg Dox treated group and the control group (p > 0.5). But when the mice were treated with 2 mg/kg free Dox, significant reduction in tumor volume and tumor weight was observed. Mice treated with 1 mg/kg LDL-Dox exhibited a significant decrease in tumor size and weight when compared with that of the control group and 1 mg/kg Dox treated group (p < 0.05 in all comparisons). This result showed that the antiproliferative effect on the growth of R-HepG2 cells in the tumor bearing nude mice of 1 mg/kg LDL-Dox was greater than that of 1 mg/kg Dox treatment and was similar to that of 2 mg/kg Dox treatment. Histological studies of heart of nude mice bearing R-HepG2 cells treated with Dox and LDL-Dox Fig. 7A shows a typical heart section from tumor bearing nude mice in which only saline was injected. In this heart section of control group, the pattern of myocardial muscle alignment and the location of muscle cell nuclei could be observed. It was found that the myocardial filaments were well organized, smooth and tightly packed. However, when the mice were treated with 1 mg/kg or 2 mg/kg of Dox, the section showed that the organization of myocardial filaments was disrupted and vacuolization was found when compared with control groups (Fig. 7B and 7C). In Fig. 7D, a heart section from 1 mg/
Fig. 5. Effect of Dox and LDL-Dox treatment on tumor size in R-HepG2-bearing nude mice. Nude mice were inoculated with 8 106 of R-HepG2 cells subcutaneously on the shoulders. After 2 days of inoculation, LDL-Dox and Dox at a dose of 1 mg/kg as well as Dox at a dose of 2 mg/kg was injected intravenously on every other day. The sizes of tumor were measured after the end of the treatment (i.e. after 4 weeks of treatment). *: p < 0.001 compared with control group, **: P < 0.005 compared with 1 mg/kg Dox treated group, # : P < 0.001 compared with 1 mg/kg Dox treated group. The result is presented as mean F S.D. n = 5 for each group.
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Fig. 6. Effect of Dox and LDL-Dox treatment on tumor weight in R-HepG2-bearing nude mice. Nude mice were inoculated with 8 106 of R-HepG2 cells subcutaneously on the shoulders. After 2 days of inoculation, LDL-Dox and Dox at a dose of 1 mg/kg as well as Dox at dose of 2 mg/kg Dox was injected intravenously on every other days. After 4 weeks of treatment, mice were anesthetized with ether and the tumors were removed and weighed *: p < 0.005 compared with control group, #: p < 0.05 compared with 1 mg/kg Dox-treated group. The result is presented as mean F S.D. n = 5 for each group.
Fig. 7. Histolograms of heart section of R-HepG2-bearing nude mice treated with Dox and LDL-Dox. Nude mice were injected subcutaneously with 8 106 of human resistant hepatoma R-HepG2 cells in the shoulder. After two days of inoculation, saline or drugs were injected intravenously (i.v.) on every other day. After 4 weeks treatment, the mice were sacrificed by cervical dislocation and the hearts were quickly removed for histological slide preparation. The ventricular wall portion was stained. The microscopic analysis was performed at magnification: 2.5 40 1 4 = 400 . (A) control (saline treatment), (B) 1 mg/kg Dox treatment, (C) 2 mg/kg Dox treatment, (D) 1 mg/kg LDL-Dox treatment.
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Fig. 8. Measurement of activities of plasma lactate dehydrogenase (LDH, n) and creatine kinase (CK, 5) in R-HepG2 tumorbearing nude mice after Dox or LDL-Dox treatments. *: p < 0.05 compared with control group, **: p < 0.05 compared with 1 mg/kg Dox treated group and 2 mg/kg Dox treated group. The result is presented as mean F S.D. n = 5 for each group.
kg LDL-Dox treated mice was shown. There was no significant disruption of myocardial filaments and vacuolization which could be found on this section when compared with control group. Activities of Plasma Lactate dehydrogenase (LDH) and Plasma Creatine kinase (CK) in R-HepG2 cellsbearing nude mice after Dox and LDL-Dox treatments Fig. 8 shows that plasma activities of LDH and CK in the Dox-treated group were higher than both control and LDL-Dox group. Moreover, plasma LDH and CK activities in LDL-Dox treated group were similar to that in the control group. This result indicated that no heart damage was observed in LDL-Dox treated group. In addition, the level of heart damage induced by Dox was higher than that by LDL-Dox.
Discussion It was widely reported that the expression level of LDL-receptors on tumor cells was higher than that on normal cells as evidenced by the finding that more LDL would be taken up in tumor cells than that in normal cells for membrane synthesis [1–4]. Moreover, cancer patients are usually found to suffer from hypocholesterolemia and hypolipoproteinemia [1]. LDL is therefore proposed as a target carrier to deliver the anticancer drug to the target tumor cells [1–4]. This strategy may increase the selectivity of the anticancer drugs to the cancer cells and reduce the adverse side effects to the normal cells. Actually, in the previous report, we found that LDL-Dox could be targeted to HepG2 cells in nude mice without causing adverse damaging effect on the heart of the host [13]. The results from the quantitative analyses in the present study on the accumulation of Dox and LDLDox in multidrug resistant R-HepG2 cells show that the amount of LDL-Dox accumulated in the cells was higher than that of free Dox (Figs. 1 and 2). When examining the cytotoxic effect of Dox and LDLDox on R-HepG2 cells, the results show that the effects of both drugs were time-dependent and dose-
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dependent. However, the cytotoxicity of LDL-Dox on R-HepG2 cells was higher when compared with that of Dox (Figs. 3 and 4). One of the probable reasons that LDL-Dox is able to circumvent the resistant cells may be because the diameter of LDL is 22 nm [19], which is bigger than the pore of P-glycoprotein whose diameter is 5 nm, so it is difficult for P-glycoprotein to bind LDL-Dox and extrude it from the cells. It was crucial to demonstrate that LDL-Dox could work as a target carrier and exhibit a better selectivity than free Dox when treating R-HepG2 cells bearing nude mice. Results of Figs. 5 and 6 on the tumor size and tumor weight in R-HepG2 cells bearing nude mice show that the tumor size and tumor weight in 1 mg/kg Dox-treated group was similar to that in the control group. But the tumor size and tumor weight in 2 mg/kg Dox-treated group was lower than that in control group. These results show that in R-HepG2 cells bearing nude mice, the lower concentration of Dox did not exhibit any antitumor effect in the R-HepG2 cells. This may be due to extrusion of Dox by the action of P-glycoprotein. In addition, the tumor size and tumor weight in 1 mg/kg LDL-Dox treated group was significantly lower than that in control group and 1 mg/kg Dox-treated group. These results imply that the anti-proliferative effect of 1 mg/kg LDL-Dox treatment was higher than that of 1 mg/kg Dox treatment. Moreover, the size and weight of tumor in 1 mg/kg LDL-Dox treated group was similar to those in 2 mg/kg Dox treated group. Since these dosages of LDL-Dox and Dox were administered 2 days after implantation of tumor cells into the nude mice, it is reasonable to believe that the decreases of the sizes and weights of tumors after drug treatments should result from the reductions of the grown tumors in the animals. The antitumor effect of LDL-Dox was higher than that of free Dox and implies that after Dox was coupled into LDL, the LDL-Dox so formed was not easy to be bound and extruded by the P-glycoprotein because of the larger size of LDL than the pore size of P-glycoprotein. These results also demonstrate that the targeted effect of LDL-Dox to the tumor cells because the lower dosage of LDL-Dox could exhibit the same antitumor effect on the R-HepG2 cells bearing nude mice treated at higher dosage of free Dox. The major side effects of Dox in patients are cardiomyopathy and congestive heart failure [20]. The results of Fig. 7 on histological studies of heart section show that in the nude mice bearing R-HepG2 cells which were only injected with normal saline (control group), the myocardial filament were well organized and tightly packed after 4 weeks treatment. However, in the Dox treated groups, including treatment of 1 mg/kg and 2 mg/kg free Dox, the vacuolization, dilation and disruption of myofibrils arrangement in the heart sections could be observed in the host when compared to the control group. These results show that free Dox could induce cardiac toxicity. In the contrast, results of LDL-Dox treated mice show that there was no apparent damages in the heart section when compared to the control group. Thus these results imply that 1 mg/kg LDL-Dox treatment exerted no cardiac toxicity and exhibited good anti-proliferative effect on tumor cells in the host. The studies on plasma lactate dehydrogenase (LDH) activity and creatine kinase (CK) activity in nude mice would also demonstrate the level of heart damage induced by different kind of treatments (Fig. 8). It was because when heart damage took place, the level of plasma LDH and CK would significantly increase. After the tumor bearing mice were treated with free Dox, at 1 mg/kg and 2 mg/kg of Dox, the plasma LDH activity in both treated groups were higher when compared with that of the control group. In the contrast, the tumor bearing mice treated with 1 mg/kg LDL-Dox exhibited a similar plasma LDH activity to that in the control group. This result suggests that the LDL-Dox-induced heart damage was much lower than that by free Dox. These results in enzymatic analyses are consistent to those obtained from the histological studies of heart section.
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The aforesaid results in animal studies give more solid evidence on the reduction of the cardiac toxic side effect by using LDL as Dox carrier. In addition, they show that lower dose of LDL-Dox could have the similar anti-proliferative effect of high dose of free Dox in R-HepG2 cells bearing mice in vivo, that means LDL-Dox could circumvent multidrug resistance in these resistant tumor cells. References [1] Alexopoulos CG, Blatsios B, Avgerinos A. Serum lipids and lipoprotein disorders in cancer patients. Cancer 1987;60:3065 – 70. [2] Ho YK, Smith RG, Brown MS, Goldstein JL. Low density lipoprotein (LDL) receptor activity in human acute myelogenous leukemia cells. Blood 1978;52:1099 – 114. [3] Klock JC, Pieprzyk JK. Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils. Journal of Lipid Research 1979;20:908 – 11. [4] Nakagawa T, Ueyama Y, Nozaki S, Yamashita S, Menju M, Funahashi T, Takemura KK, Kubo M, Tokunaga K, Tanaka T, Yagi M, Matsuzawa Y. Marked Hypocholesterolemia in a Case with Adrenal Adenoma — Enhanced Catabolism of Low Density Lipoprotein (LDL) via the LDL Receptors of Tumor Cells. Journal of Clinical Endocrinology and Metabolism 1994;80:92 – 6. [5] Arcamone F, Cassinelli G, Fantini G, Grein A, Orezzi P, Pol C, Spalla C. Adriamycin, 14-hydroxydaunomycin, a new antitumor antibiotic from S. peucetius var. caesius. Biotechnology and Bioengineering 1969;11:1101 – 10. [6] Fogleson PD, Reckord C, Swink S. Doxorubicin inhibits human DNA topoisomerase I. Cancer Chemotherapy and Pharmacology 1992;30:123 – 5. [7] Tewey KM, Rowe TC, Yang L. Adriamycin-induced DNA damage mediated by mammalian DNA topisomerase II. Science 1984;226:466 – 8. [8] Bachur NR, Yu F, Johnson R. Helicase inhibition by anthracycline anticancer agents. Molecular Pharmacology 1992;41:993 – 8. [9] Koseki Y, Israel M, Sweatman TW. Inhibitory effects of immobilized adriamycin on cell growth and thymidine uptake in human CEM leukemic lymphocytes. Journal of Cell Pharmacology 1991;2:171 – 8. [10] Bachur NR, Gordon SL, Gee MW. Anthracycline antibiotic augmentation of microsomal electron transport and free radical formation. Molecular Pharmacology 1977;13:901 – 10. [11] Roninson IB, Chin JE, Choi K. Isolation of human mdr DNA sequences amplified in multidrug-resistance KB carcinoma cells. Proceedings of National Academy of Sciences (U.S.A.) 1986;83:4538 – 42. [12] Gottesman MM, Pastan I. The multidrug-transporter: a double-edged sword. The Journal of Biological Chemistry 1988;263:12163 – 6. [13] Chu ACY, Tsang SY, Lo EHK, Fung KP. Low density lipoprotein as a targeted carrier for doxorubicin in nude mice bearing tumor hepatoma HepG2 cells. Life Sciences 2001;70:591 – 601. [14] Chan JYW, Chu ACY, Fung KP. Inhibition of P-glycoprotein expression and reversal of drug resistance of human hepatoma HepG2 cells by multidrug resistance gene (mdr1) antisense RNA. Life Sciences 2000;67:2117 – 24. [15] Rudling MJ, Collins VP, Peterson CO. Delivery of aclacinomycin A to human glioma cells in vitro by the low-density lipoprotein pathway. Cancer Research 1983;43:4600 – 5. [16] Iwanik MJ, Shaw KV, Ledwith BJ, Yanovich S, Shaw JM. Preparation of interaction of a low-density lipoprotein: daunomycin complex with P388 leukemic cells. Cancer Research 1984;44:1206 – 15. [17] Lew YS, Brown SL, Griffin RJ, Song CW, Kim JH. Arsenic trioxide causes selective necrosis in solid murine tumors by vascular shutdown. Cancer Research 1999;59:6033 – 7. [18] Olson HM, Young DM, Prieur DJ, Leroy AF, Reagar RL. Electrolyte and morphologic alterations of myocardium in adriamycin-treated rabbits. The American Journal of Pathology 1974;77:439 – 54. [19] Krieger M, Smith LC, Anderson RGW, Goldstein JL, Kao YJ, Pownall HJ, Gotto AMJ, Brown MS. Reconstituted low density lipoprotein: a vehicle for the delivery of hydrophobic fluorescent probe to cells. Journal of Supramolecular Structure 1979;10:467 – 78. [20] Singal PK, Iliskovic N, Li T, Kumar D. Adriamycin cardiomyopathy: pathophysiology and prevention. The FASEB Journal 1997;11:931 – 6.
Circumvention of multidrug resistance and reduction of cardiotoxicity of doxorubicin in vivo by coupling it $ with low density lipoprotein Elka H.K. Lo a, Vincent E.L. Ooi b, K.P. Fung a,* a
Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China b Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong, China Received 12 December 2001; accepted 24 July 2002
Abstract Doxorubicin (Dox) was coupled into human low density lipoprotein (LDL) to form a complex LDL-Dox. In in vitro studies, the accumulation of LDL-Dox in human resistant hepatoma (R-HepG2) cells was found to be higher than that of free Dox in the cells, resulting in an increase of the cytotoxic effect on the cells. Moreover, in in vivo studies, under the same dosage of drugs (1 mg/kg), the anti-proliferative effect on the tumor cells of LDL-Dox in nude mice bearing R-HepG2 cells was higher than that of free Dox as evidenced by the larger reduction in tumor volumes and tumor weights in LDL-Dox treated group. Histological studies showed that LDL-Dox treatment did not cause any heart damage when compared with the control group. In contrast, Dox treatment caused disruption and vacuolization of myocardial filament. Plasma lactate dehydrogenase activity and plasma creatine kinase activity in nude mice bearing R-HepG2 cells were found to be elevated in the Dox-treated group but remained unchanged in LDL-Dox-treated group. The present studies indicate that when Dox is coupled with LDL, the multidrug resistance can be circumvented and the cardiotoxicity can be reduced. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Doxorubicin; Low density lipoprotein; Human resistant hepatoma cells
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This work was supported by an earmarked grant from Research Grants Council (CUHK4060/97M, Hong Kong, China). * Corresponding author. Tel.: +852-2603-6873; fax: +852-2603-5123. E-mail address: [email protected] (K.P. Fung).
0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 1 8 0 - X
678
E.H.K. Lo et al. / Life Sciences 72 (2002) 677–687
Introduction Antitumoral drugs always exhibit adverse side effects on normal cells because of the lack of selectivity. Moreover, the drug resistance is usually a problem in chemotherapy. It has been an urgent need to develop a target carrier for antitumoral drugs in order to increase the specificity of drugs and the chance of circumvention of multidrug resistance on tumor cells and to decrease the drugs’ toxicities to normal cells. Low density lipoprotein (LDL) was proposed as a target carrier to deliver antitumoral drugs because many studies showed that the cancer cells have a higher number of low density lipoprotein receptors (LDL-R) than the normal cells [1–4]. Doxorubicin (Dox) is one of the prototypes of anthracycline anticancer antibiotic with anti-tumoral activity which is isolated from strains of Streptomyces peucetius [5]. Dox is currently used widely in treating different tumors. The multiple potential mechanistic actions of Dox include the inhibition of activities of DNA topoisomerase I [6] and II [7], inhibition of helicases [8], alteration of membrane structure and function [9] and generation of free radicals [10]. Since the hearts contain high content of oxygen, a large amount of free radicals are produced by Dox causing cadiotoxicity which is the main adverse side effect of Dox, especially during prolonged treatment in the patients. Development of resistance to the multiple antitumoral drugs in cancer cells after prolonged treatment is another major problem of chemotherapy. One of the reasons of multidrug resistance is the overexpression of P-glycoprotein (P-gp) [11]. P-gp can exert active drug efflux out of resistant cells in order to reduce the accumulation of various types of antitumoral drugs and thus preventing them from reaching their cellular targets [12]. In previous studies, we found that LDL could serve as a target carrier for Dox to kill tumor cells in nude mice bearing human hepatoma HepG2 cells [13]. In the present study, we further investigate the cytotoxicity of LDL-Dox on R-HepG2 cells and to examine whether LDL-Dox can circumvent the drug resistance in this resistant cell line. Moreover, we have made use of the R-HepG2 tumor cells bearing nude mice model to investigate the anti-proliferative effect of LDL-Dox on tumor cells and its cardiotoxicity when compared with free Dox.
Methods Cell culture The human resistant hepatoma cell line (R-HepG2) was developed in our laboratory by incubating HepG2 cells in stepwise increasing concentrations of doxorubicin [14]. Treatments were repeated until the cell grew at 1.2 AM doxorubicin. R-HepG2 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 1.2 AM doxorubicin. The resulting resistant cells were found to express high content of P-gp by Northern and Western analysis, and exhibit resistance to Dox [14]. Formation of LDL-Dox complex The method reported in Refs. [15,16] was adopted with minor modifications [13]. Ten milligram of doxorubicin was dissolved in 1 ml of autoclaved double distilled water. LDL was mixed with Dox at the
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ratio of 10:1 (w/w). The mixture was incubated in shaking air-bath at 37 jC for 24 hours in dark. The mixture was then loaded onto gel filtration column with G25 Sephadex to separate the free Dox from the LDL-Dox complex. Fractions of 0.5 ml were collected. The LDL-Dox complex so obtained was sterilized by passage through a 0.45 Am acetate millipore filter. After Dox was coupled into LDL, its concentration in the complex was measured. Twenty microlitre of LDL-Dox complex was added to 780 Al of acidified isopropanol. A standard curve of the concentration of doxorubicin in acidified isopropanol versus the absorbance (O.D.) at wavelength 480 nm was obtained. From the curve, the concentration of LDL-Dox was determined. Determination of the accumulation level of Dox in cultured R-HepG2 cells after Dox and LDL-Dox treatments by flow cytometry and confocal laser scanning microscope R-HepG2 cells were seeded in a 6-well plate and incubated at 37 jC, 5% CO2 overnight. The cells were washed twice with PBS. The medium was replaced by RPMI 1640 medium with 10% lipoprotein deficient fetal bovine serum (LPDS) and incubated for further 48 hours at 37 jC, 5% CO2. After that, the medium was removed and the cells were washed twice with PBS. Then, 2 ml of 1 AM of LDL-Dox or Dox in RPMI with 10% LPDS were added and incubated for 1 hour at 37 jC. The adhered cells were trypsinised and the fluorescence intensities of Dox in the cells were analyzed by flow cytometry with FAC Sort (BECTON DICKINSON). Forward and side scatters were used to establish size gates and exclude debris from the analysis. The excitation wavelength was 488 nm while the emitted fluorescence was collected at 585 nm. For confocal microscopic analysis, R-HepG2 cells were first seeded on cover glasses. After the same treatment protocol, cell images were acquired on Multiprobe 2001 from Molecular Dynamics. An excitation filter with 488 nm wavelength and a long-pass emission filter of 510 nm were used. After scanning, images were processed by an image analysis software [13]. Determination of survival of cultured R-HepG2 cells after Dox and LDL-Dox treatments The survival of R-HepG2 cells was determined by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay as described before [13]. The results were presented as mean of percentage of cell survival F S.D. The percentage of cell survival was calculated by dividing the absorbance of treated cells and the absorbance of untreated cell multiplied by 100. Effects of Dox and LDL-Dox on tumor growth in nude mice bearing R-HepG2 cells Nu/nu nude mice (with body weight of 20–25 g and aged 6–8 weeks) were kept in autoclaved cage with polyester fiber filters to avoid contact with the pathogens. All the animal diet (PICO LAB@ Rodent Diet) and tape water were autoclaved before feeding to nude mice ad libitum. Suspension of 8 106 of human resistant hepatoma R-HepG2 cells were injected subcutaneously (s.c.) into the anterior part of the shoulder of the nude mice. Drug treatments were started 2 days after implantation of cells. The mice were then randomly divided into treatment group and control group with 5 mice in each group. Free Dox at dosage 1 mg/kg and 2 mg/kg as well as LDL-Dox at dosage 1 mg/kg in saline were injected into the mice intravenously on 1 injection per 2 days schedule. The injections were given for 4 weeks. Tumor volume was measured by Vernier caliper weekly and tumor volumes
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were calculated according to the formula [17]: Tumor volume (mm3) = a/2 b/2 h k, where a, b and h are the minor and major dimensions and height of the tumor, respectively, k = 3.1416. After the treatment, the nude mice were anesthetized with diethyl ether and the weights of the tumors were recorded. Investigation of the myocardial injury in nude mice after Dox and LDL-Dox treatments Myocardial injury was assessed by measuring the leakage of lactate dehydrogenase (LDH) and creatine kinase (CK) from the heart to the blood. After treatment for four weeks, the tumor-bearing nude mice were anesthetized and the blood was collected in heparinized tube. Plasma LDH and CK activity were then assayed by using LDH assay kit and CK assay kit (SIGMA), respectively. Histological analysis of the hearts in nude mice after Dox and LDL-Dox treatments Another group of tumor-bearing nude mice were sacrificed by cervical dislocation on the next day after the last injection of drug. Hearts were removed immediately and the sections were prepared and stained by standard histological method [18]. The sections were observed under light microscope (OLYMPUS) by different combinations of magnification values of the eye pieces and objective lens of the microscope. Micrographs of the heart sections under microscope were taken for record and comparison [18]. Statistics Student’s t test was used to compare the data obtained in Dox-treated and LDL-Dox-treated groups.
Results The comparison of the accumulation of Dox and LDL-Dox in R-HepG2 cells The accumulation of Dox and LDL-Dox in R-HepG2 cells were determined by flow cytometry. As shown in Fig. 1, there was no significant difference in fluorescent intensity of R-HepG2 cells when incubated with or without Dox. In contrast, an increase in fluorescent intensity was observed in cells treated with LDL-Dox than that treated with free Dox. Fig. 2 shows the result of confocal laser scanning microscopic images. It was found that R-HepG2 cells treated with LDL-Dox exhibited a higher degree of fluorescent intensity than that with free Dox. Moreover, the accumulated free Dox was found to be located mainly near the cell membrane. This result suggests that the intracellular accumulation of LDL-Dox was higher than that of free Dox in R-HepG2 cells. Comparison of cytotoxicity of Dox and LDL-Dox on R-HepG2 cells The percentage cell survival after Dox or LDL-Dox treatment was determined by MTT assay (Figs. 3 and 4). For 24 hours incubation, the IC50 of Dox and LDL-Dox were found to be 368 AM and 48
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Fig. 1. The quantitative analysis of Dox and LDL-Dox accumulated in R-HepG2 cells. The accumulation of Dox and LDL-Dox after 1 hour incubation were measured by flow cytometry.
AM, respectively. When the incubation time was prolonged from 24 hours to 48 hours, the IC50 of Dox and LDL-Dox were found to be 300 AM and 15 AM, respectively. After 72 hours incubation, IC50 of Dox and LDL-Dox were found to be 240 AM and 7 AM, respectively. These results showed
Fig. 2. The confocal laser scanning microscopic analysis on the accumulated level of Dox and LDL-Dox in R-HepG2 cells after 1 hour incubation. Upper panel: Dox-treated R-HepG2 cells; Lower panel: LDL-Dox-treated R-HepG2 cells.
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Fig. 3. The cytotoxicity of Dox on R-HepG2 cells after 24, 48 and 72 hours incubation. The percentage of cell survival was analyzed by MTT assay. x—x, 24 hours; n—n, 48 hours; E—E, 72 hours. The result is presented as mean F S.D. (n = 5).
that as the concentration of Dox and LDL-Dox increased, the numbers of cell viability were decreased, i.e. the percentage of cell survival was dose- and time-dependent for both free Dox and LDL-Dox treatments. In addition, the IC50 of LDL-Dox was lower than that of free Dox at different time points. This result indicated that after Dox was coupled into LDL, its cytotoxic effect in RHepG2 cells could be enhanced i.e. the multidrug resistance of R-HepG2 cells could be circumvented.
Fig. 4. The cytotoxicity of LDL-Dox on R-HepG2 cells after 24, 48 and 72 hours incubation. The percentage of cell survival was analyzed by MTT assay. x—x, 24 hours; n—n, 48 hours; E—E, 72 hours. The result is presented as mean F S.D. (n = 5).
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Treatment of tumor-bearing nude mice with free Dox and LDL-Dox The anti-proliferative effect of Dox and LDL-Dox on the growth of R-HepG2 cells in tumor bearing nude mice was investigated by measuring the tumor volume and tumor weight in the mice after treatment for 4 weeks. Fig. 5 and Fig. 6 showed that there was no significant difference in tumor volume and tumor weight between 1 mg/kg Dox treated group and the control group (p > 0.5). But when the mice were treated with 2 mg/kg free Dox, significant reduction in tumor volume and tumor weight was observed. Mice treated with 1 mg/kg LDL-Dox exhibited a significant decrease in tumor size and weight when compared with that of the control group and 1 mg/kg Dox treated group (p < 0.05 in all comparisons). This result showed that the antiproliferative effect on the growth of R-HepG2 cells in the tumor bearing nude mice of 1 mg/kg LDL-Dox was greater than that of 1 mg/kg Dox treatment and was similar to that of 2 mg/kg Dox treatment. Histological studies of heart of nude mice bearing R-HepG2 cells treated with Dox and LDL-Dox Fig. 7A shows a typical heart section from tumor bearing nude mice in which only saline was injected. In this heart section of control group, the pattern of myocardial muscle alignment and the location of muscle cell nuclei could be observed. It was found that the myocardial filaments were well organized, smooth and tightly packed. However, when the mice were treated with 1 mg/kg or 2 mg/kg of Dox, the section showed that the organization of myocardial filaments was disrupted and vacuolization was found when compared with control groups (Fig. 7B and 7C). In Fig. 7D, a heart section from 1 mg/
Fig. 5. Effect of Dox and LDL-Dox treatment on tumor size in R-HepG2-bearing nude mice. Nude mice were inoculated with 8 106 of R-HepG2 cells subcutaneously on the shoulders. After 2 days of inoculation, LDL-Dox and Dox at a dose of 1 mg/kg as well as Dox at a dose of 2 mg/kg was injected intravenously on every other day. The sizes of tumor were measured after the end of the treatment (i.e. after 4 weeks of treatment). *: p < 0.001 compared with control group, **: P < 0.005 compared with 1 mg/kg Dox treated group, # : P < 0.001 compared with 1 mg/kg Dox treated group. The result is presented as mean F S.D. n = 5 for each group.
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Fig. 6. Effect of Dox and LDL-Dox treatment on tumor weight in R-HepG2-bearing nude mice. Nude mice were inoculated with 8 106 of R-HepG2 cells subcutaneously on the shoulders. After 2 days of inoculation, LDL-Dox and Dox at a dose of 1 mg/kg as well as Dox at dose of 2 mg/kg Dox was injected intravenously on every other days. After 4 weeks of treatment, mice were anesthetized with ether and the tumors were removed and weighed *: p < 0.005 compared with control group, #: p < 0.05 compared with 1 mg/kg Dox-treated group. The result is presented as mean F S.D. n = 5 for each group.
Fig. 7. Histolograms of heart section of R-HepG2-bearing nude mice treated with Dox and LDL-Dox. Nude mice were injected subcutaneously with 8 106 of human resistant hepatoma R-HepG2 cells in the shoulder. After two days of inoculation, saline or drugs were injected intravenously (i.v.) on every other day. After 4 weeks treatment, the mice were sacrificed by cervical dislocation and the hearts were quickly removed for histological slide preparation. The ventricular wall portion was stained. The microscopic analysis was performed at magnification: 2.5 40 1 4 = 400 . (A) control (saline treatment), (B) 1 mg/kg Dox treatment, (C) 2 mg/kg Dox treatment, (D) 1 mg/kg LDL-Dox treatment.
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Fig. 8. Measurement of activities of plasma lactate dehydrogenase (LDH, n) and creatine kinase (CK, 5) in R-HepG2 tumorbearing nude mice after Dox or LDL-Dox treatments. *: p < 0.05 compared with control group, **: p < 0.05 compared with 1 mg/kg Dox treated group and 2 mg/kg Dox treated group. The result is presented as mean F S.D. n = 5 for each group.
kg LDL-Dox treated mice was shown. There was no significant disruption of myocardial filaments and vacuolization which could be found on this section when compared with control group. Activities of Plasma Lactate dehydrogenase (LDH) and Plasma Creatine kinase (CK) in R-HepG2 cellsbearing nude mice after Dox and LDL-Dox treatments Fig. 8 shows that plasma activities of LDH and CK in the Dox-treated group were higher than both control and LDL-Dox group. Moreover, plasma LDH and CK activities in LDL-Dox treated group were similar to that in the control group. This result indicated that no heart damage was observed in LDL-Dox treated group. In addition, the level of heart damage induced by Dox was higher than that by LDL-Dox.
Discussion It was widely reported that the expression level of LDL-receptors on tumor cells was higher than that on normal cells as evidenced by the finding that more LDL would be taken up in tumor cells than that in normal cells for membrane synthesis [1–4]. Moreover, cancer patients are usually found to suffer from hypocholesterolemia and hypolipoproteinemia [1]. LDL is therefore proposed as a target carrier to deliver the anticancer drug to the target tumor cells [1–4]. This strategy may increase the selectivity of the anticancer drugs to the cancer cells and reduce the adverse side effects to the normal cells. Actually, in the previous report, we found that LDL-Dox could be targeted to HepG2 cells in nude mice without causing adverse damaging effect on the heart of the host [13]. The results from the quantitative analyses in the present study on the accumulation of Dox and LDLDox in multidrug resistant R-HepG2 cells show that the amount of LDL-Dox accumulated in the cells was higher than that of free Dox (Figs. 1 and 2). When examining the cytotoxic effect of Dox and LDLDox on R-HepG2 cells, the results show that the effects of both drugs were time-dependent and dose-
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dependent. However, the cytotoxicity of LDL-Dox on R-HepG2 cells was higher when compared with that of Dox (Figs. 3 and 4). One of the probable reasons that LDL-Dox is able to circumvent the resistant cells may be because the diameter of LDL is 22 nm [19], which is bigger than the pore of P-glycoprotein whose diameter is 5 nm, so it is difficult for P-glycoprotein to bind LDL-Dox and extrude it from the cells. It was crucial to demonstrate that LDL-Dox could work as a target carrier and exhibit a better selectivity than free Dox when treating R-HepG2 cells bearing nude mice. Results of Figs. 5 and 6 on the tumor size and tumor weight in R-HepG2 cells bearing nude mice show that the tumor size and tumor weight in 1 mg/kg Dox-treated group was similar to that in the control group. But the tumor size and tumor weight in 2 mg/kg Dox-treated group was lower than that in control group. These results show that in R-HepG2 cells bearing nude mice, the lower concentration of Dox did not exhibit any antitumor effect in the R-HepG2 cells. This may be due to extrusion of Dox by the action of P-glycoprotein. In addition, the tumor size and tumor weight in 1 mg/kg LDL-Dox treated group was significantly lower than that in control group and 1 mg/kg Dox-treated group. These results imply that the anti-proliferative effect of 1 mg/kg LDL-Dox treatment was higher than that of 1 mg/kg Dox treatment. Moreover, the size and weight of tumor in 1 mg/kg LDL-Dox treated group was similar to those in 2 mg/kg Dox treated group. Since these dosages of LDL-Dox and Dox were administered 2 days after implantation of tumor cells into the nude mice, it is reasonable to believe that the decreases of the sizes and weights of tumors after drug treatments should result from the reductions of the grown tumors in the animals. The antitumor effect of LDL-Dox was higher than that of free Dox and implies that after Dox was coupled into LDL, the LDL-Dox so formed was not easy to be bound and extruded by the P-glycoprotein because of the larger size of LDL than the pore size of P-glycoprotein. These results also demonstrate that the targeted effect of LDL-Dox to the tumor cells because the lower dosage of LDL-Dox could exhibit the same antitumor effect on the R-HepG2 cells bearing nude mice treated at higher dosage of free Dox. The major side effects of Dox in patients are cardiomyopathy and congestive heart failure [20]. The results of Fig. 7 on histological studies of heart section show that in the nude mice bearing R-HepG2 cells which were only injected with normal saline (control group), the myocardial filament were well organized and tightly packed after 4 weeks treatment. However, in the Dox treated groups, including treatment of 1 mg/kg and 2 mg/kg free Dox, the vacuolization, dilation and disruption of myofibrils arrangement in the heart sections could be observed in the host when compared to the control group. These results show that free Dox could induce cardiac toxicity. In the contrast, results of LDL-Dox treated mice show that there was no apparent damages in the heart section when compared to the control group. Thus these results imply that 1 mg/kg LDL-Dox treatment exerted no cardiac toxicity and exhibited good anti-proliferative effect on tumor cells in the host. The studies on plasma lactate dehydrogenase (LDH) activity and creatine kinase (CK) activity in nude mice would also demonstrate the level of heart damage induced by different kind of treatments (Fig. 8). It was because when heart damage took place, the level of plasma LDH and CK would significantly increase. After the tumor bearing mice were treated with free Dox, at 1 mg/kg and 2 mg/kg of Dox, the plasma LDH activity in both treated groups were higher when compared with that of the control group. In the contrast, the tumor bearing mice treated with 1 mg/kg LDL-Dox exhibited a similar plasma LDH activity to that in the control group. This result suggests that the LDL-Dox-induced heart damage was much lower than that by free Dox. These results in enzymatic analyses are consistent to those obtained from the histological studies of heart section.
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The aforesaid results in animal studies give more solid evidence on the reduction of the cardiac toxic side effect by using LDL as Dox carrier. In addition, they show that lower dose of LDL-Dox could have the similar anti-proliferative effect of high dose of free Dox in R-HepG2 cells bearing mice in vivo, that means LDL-Dox could circumvent multidrug resistance in these resistant tumor cells. References [1] Alexopoulos CG, Blatsios B, Avgerinos A. Serum lipids and lipoprotein disorders in cancer patients. Cancer 1987;60:3065 – 70. [2] Ho YK, Smith RG, Brown MS, Goldstein JL. Low density lipoprotein (LDL) receptor activity in human acute myelogenous leukemia cells. Blood 1978;52:1099 – 114. [3] Klock JC, Pieprzyk JK. Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils. Journal of Lipid Research 1979;20:908 – 11. [4] Nakagawa T, Ueyama Y, Nozaki S, Yamashita S, Menju M, Funahashi T, Takemura KK, Kubo M, Tokunaga K, Tanaka T, Yagi M, Matsuzawa Y. Marked Hypocholesterolemia in a Case with Adrenal Adenoma — Enhanced Catabolism of Low Density Lipoprotein (LDL) via the LDL Receptors of Tumor Cells. Journal of Clinical Endocrinology and Metabolism 1994;80:92 – 6. [5] Arcamone F, Cassinelli G, Fantini G, Grein A, Orezzi P, Pol C, Spalla C. Adriamycin, 14-hydroxydaunomycin, a new antitumor antibiotic from S. peucetius var. caesius. Biotechnology and Bioengineering 1969;11:1101 – 10. [6] Fogleson PD, Reckord C, Swink S. Doxorubicin inhibits human DNA topoisomerase I. Cancer Chemotherapy and Pharmacology 1992;30:123 – 5. [7] Tewey KM, Rowe TC, Yang L. Adriamycin-induced DNA damage mediated by mammalian DNA topisomerase II. Science 1984;226:466 – 8. [8] Bachur NR, Yu F, Johnson R. Helicase inhibition by anthracycline anticancer agents. Molecular Pharmacology 1992;41:993 – 8. [9] Koseki Y, Israel M, Sweatman TW. Inhibitory effects of immobilized adriamycin on cell growth and thymidine uptake in human CEM leukemic lymphocytes. Journal of Cell Pharmacology 1991;2:171 – 8. [10] Bachur NR, Gordon SL, Gee MW. Anthracycline antibiotic augmentation of microsomal electron transport and free radical formation. Molecular Pharmacology 1977;13:901 – 10. [11] Roninson IB, Chin JE, Choi K. Isolation of human mdr DNA sequences amplified in multidrug-resistance KB carcinoma cells. Proceedings of National Academy of Sciences (U.S.A.) 1986;83:4538 – 42. [12] Gottesman MM, Pastan I. The multidrug-transporter: a double-edged sword. The Journal of Biological Chemistry 1988;263:12163 – 6. [13] Chu ACY, Tsang SY, Lo EHK, Fung KP. Low density lipoprotein as a targeted carrier for doxorubicin in nude mice bearing tumor hepatoma HepG2 cells. Life Sciences 2001;70:591 – 601. [14] Chan JYW, Chu ACY, Fung KP. Inhibition of P-glycoprotein expression and reversal of drug resistance of human hepatoma HepG2 cells by multidrug resistance gene (mdr1) antisense RNA. Life Sciences 2000;67:2117 – 24. [15] Rudling MJ, Collins VP, Peterson CO. Delivery of aclacinomycin A to human glioma cells in vitro by the low-density lipoprotein pathway. Cancer Research 1983;43:4600 – 5. [16] Iwanik MJ, Shaw KV, Ledwith BJ, Yanovich S, Shaw JM. Preparation of interaction of a low-density lipoprotein: daunomycin complex with P388 leukemic cells. Cancer Research 1984;44:1206 – 15. [17] Lew YS, Brown SL, Griffin RJ, Song CW, Kim JH. Arsenic trioxide causes selective necrosis in solid murine tumors by vascular shutdown. Cancer Research 1999;59:6033 – 7. [18] Olson HM, Young DM, Prieur DJ, Leroy AF, Reagar RL. Electrolyte and morphologic alterations of myocardium in adriamycin-treated rabbits. The American Journal of Pathology 1974;77:439 – 54. [19] Krieger M, Smith LC, Anderson RGW, Goldstein JL, Kao YJ, Pownall HJ, Gotto AMJ, Brown MS. Reconstituted low density lipoprotein: a vehicle for the delivery of hydrophobic fluorescent probe to cells. Journal of Supramolecular Structure 1979;10:467 – 78. [20] Singal PK, Iliskovic N, Li T, Kumar D. Adriamycin cardiomyopathy: pathophysiology and prevention. The FASEB Journal 1997;11:931 – 6.