ALPHA-FETOPROTEIN-MEDIATED TARGETING—A NEW STRATEGY TO OVERCOME MULTIDRUG RESISTANCE OF TUMOUR CELLSIN VITRO

Cell Biology International, 1997, Vol. 21, No. 12, 793–799 Article No. cb980201

ALPHA-FETOPROTEIN-MEDIATED TARGETING—A NEW STRATEGY TO OVERCOME MULTIDRUG RESISTANCE OF TUMOUR CELLS IN VITRO ELIZAVETA YU. MOSKALEVA, GALINA A. POSYPANOVA, IGOR I. SHMYREV, ALLA V. RODINA, EKATERINA L. MUIZHNEK, EUGENE S. SEVERIN, VALERY YU. KATUKOV, YURY M. LUZHKOV and SERGEI E. SEVERIN* Moscow Research Institute of Medical Ecology, Sympheropolsky blvd.8, Moscow, 113149, Russia Accepted 26 November 1997

The possibility of overcoming the multidrug resistance of human malignant cells by using doxorubicin conjugated to alpha-fetoprotein (AFP) was studied. It was shown that this type of antitumour drugs, penetrating the cell by receptor-mediated endocytosis with AFP as a vehicle, raises the sensitivity of the tumour cells that are resistant due to the expression of the multidrug resistance gene mdr1. The sensitivity of antibiotic-resistant cell lines SKVLB (a human ovarian carcinoma) and MCF-7 AdrR (a human breast carcinoma) increased by 10- and 4-fold, respectively, when AFP-conjugated doxorubicin was used. The rationale of using human AFP-antitumour drug conjugates for the development of new chemotherapeutic approaches to  1997 Academic Press cancer treatment is discussed. K: AFP; AFP-conjugates with drugs (doxorubicin); targeted delivery; multidrug resistance; P-glycoprotein; tumour cells

INTRODUCTION The major problems of cancer chemotherapy that still await their solution are low selectivity of antitumour drugs towards cancer cells and the fast development of drug resistance by the tumour cells. One of the possible solutions to the first problem is the development of better drug homing systems. Encouraging results have been obtained in this field. Design of anticancer drugs in the form of cytotoxic agents conjugated with various vector molecules, e.g. antibodies to tumour cell receptors or physiological ligands of these receptors (growth factors and oncofetal proteins), is rapidly growing in popularity (Ford and Casson, 1986; Embleton, 1987; Pastan et al., 1986; Friedman et al., 1993; Liao et al., 1995; Hoshino et al., 1995). In the latter category, alpha-fetoprotein (AFP) is considered to be most promising. This protein (mw 66,000) induces receptor-mediated endocytosis after binding to surface receptors on tumour cells (Uriel et al., 1984, 1987; Naval et al., 1985; Laborda et al., *To whom correspondence should be addressed. 1065–6995/97/120793+07 $30.00/0

1987; Torres et al., 1991; Moro et al., 1993). The level of AFP receptor expression in nonproliferating cells is very low (Torres et al., 1989). Even proliferating phytohaemagglutininstimulated peripheral blood lymphocytes expose no more than 88,000 AFP receptors per cell (Torres et al., 1989), while their number on the surface of tumour cells of various origin, including mouse mammary carcinoma, human breast cancer, neuroblastoma, lymphoma, hepatoma, adenocarcinoma, etc., varies from several hundreds to one million per cell. In particular, cells of human breast carcinoma line MCF-7 contain about 140,000 AFP-binding sites per one cell (Sarcione et al., 1983), while T-cell human lymphoma line CEM contain about one million (Torres et al., 1991). There are several reports in the current literature concerning the isolation and purification of the AFP receptor (Sarcione et al., 1983; Suzuki et al., 1992; Moro et al., 1993). It has been shown that AFP receptor complexes with AFP are often discovered during the isolation of AFP receptor. The molecular weight of the AFP receptor is approximately 65 kDa (Moro et al., 1993).  1997 Academic Press

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Electron microscopic studies revealed that in the course of endocytosis covalent conjugates of AFP with horseradish peroxidase (the latter was used for visualization of AFP) are first detected in clathrin vesicles, then in endosomes and folded membranes of the central region of the Golgi complex. After internalization the greater part of AFP is not subject to degradation but is repeatedly released into the extracellular space, i.e. AFP undergoes recycling (Naval et al., 1985; Torres et al., 1991). The possibility to use AFP as a vehicle for receptor-mediated transport of anticancer drugs has been recently demonstrated in our laboratory (Severin et al., 1995, 1996). As for the second problem, that is, the ability of malignant cells to resist cytostatic drugs, researchers and clinicians have poor grounds for optimism. The last 10–15 years have yielded much data concerning the formation of tumour cell resistance; however, no significant progress has thus far been attained on the way to the successful obviating of this problem. Several established mechanisms explain the appearance of cellular resistance (Sikic, 1993; Skovsgaard et al., 1994; Broxterman et al., 1995). The most studied phenomenon is the so-called classic multidrug resistance (MDR) (Bosmann, 1971), which implies simultaneous development of cellular resistance to various types of drugs. The main reason for MDR occurrence is activation (overexpression and amplification) of the multidrug resistance gene (mdr1). As a result of mdr1 activation, an increase in formation of plasmalemma glycoprotein Pgp (mw 170,000) occurs (Endicott and Ling, 1989; Schinkel and Borst, 1991). Pgp functions as an ATP-dependent pump, removing various antitumour drugs from the cytoplasm, thus decreasing the drug accumulation by the cell. Thus, most approaches to MDR modulation have been focused on developing pathways for inhibiting the expression and/or activity of Pgp (Germann and Harding, 1995). At present, many preparations capable of more or less reversing the MDR in vitro have been studied. These substances have different chemical structures, some block Ca2+ -channels (verapamil), some are calmodulin inhibitors, while others represent steroids, detergents, antibiotics, immunosuppressants, etc. Most of them efficiently inhibit the drug efflux function of Pgp via direct interaction with the mdr1 gene product (e.g. the cyclosporin PSC 833), and some (e.g. verapamil and cyclosporine A) represent substrates for Pgp-mediated transport (Skovsgaard et al., 1994; Broxterman

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et al., 1995; Germann and Harding, 1995). However, clinical tests of first generation MDRmodulators (verapamil, cyclosporin A) have failed to reach the desired goal due to the high toxicity of these preparations on normal tissues, resulting in a great number of important side effects. In other words, a rather high level of chemosensitizer (e.g. verapamil 1–10 ì) needed in vitro to reverse experimental MDR-levels that cannot be achieved clinically. Moreover, clinical drug resistance is most likely due to a complex interplay of many factors (e.g. pharmacological factors, hypoxia, low growth fraction of the tumour cell population, etc.). Finally, since tumour cell resistance is probably multifactorial, other mechanisms a part from increased Pgp expression will often play a role (Sikic, 1993; Bellamy and Dalton, 1994; Skovsgaard et al., 1994; Fisher and Sikic, 1995). The development of MDR modulators of the second generation, such as dexverapamil, S9788 (triazinoaminopiperidine derivative), MS-073 and MS-209 (quinoline compounds), SDZ PSC 833 (a non-immunosuppressive cyclosporin which in vitro is as potent as cyclosporin A), and some other drugs characterized by lower toxicity and their clinical tests were positive in some cases, in particular in the treatment of hematolymphatic malignancies (Fisher and Sikic, 1995; Slate et al., 1995; Wilson et al., 1995). At the same time, results of studies in patients with solid tumours have been somewhat disappointing (Bellamy and Dalton, 1994; Fisher and Sikic, 1995). This prompts the necessity of a search for entirely different approaches to the solution of the major problem, namely, how to overcome multidrug resistance. Thus, the possibility of modulation of anticancer drug transport by signal transduction pathways, involving systems such as drug transporter activity, topoisomerase II activity and the apoptotic pathway, still remains to be investigated. Many antitumour drugs possess membrane-bound activity and, consequently, can participate in such type of regulation. For example, it is possible to modulate the Pgp activity by inhibiting or activating protein kinase C in vitro (Bates et al., 1993). There are some similar data concerning tyrosine kinase inhibitors (Broxterman et al., 1995). As it has been pointed out earlier, we have obtained conjugates of anticancer drug doxorubicin (Dr) with AFP as a vehicle. We showed that these conjugates displayed cytotoxicity levels several times higher than that of free Dr, even on Dr-resistant cells, as well as a high level of selectivity towards tumour cells (Severin et al., 1995; Severin et al., 1996).

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In contrast, with free antitumour drugs penetrating tumour cells by diffusion, the mechanism of AFP-conjugate delivery into the cell is via receptormediated transport. It would be reasonable to suppose that in this case the conjugated drugs will not be removed from the cells by means of Pgpmediated efflux and that, consequently, their application will raise the sensitivity of resistant cells. The aim of the present work was to test this hypothesis experimentally. MATERIALS AND METHODS Purification of AFP AFP was isolated from fresh female retroplacental serum by immunoaffinity chromatography (Severin et al., 1995). The protein was lyophilized and stored at 4C. AFP concentration was determined by radial immunodiffusion according to Manchini (measuring the precipitate diameters and using the accompanying Table of Reference Values, Behring kit) and enzyme immunoassay kit (Hoffmann, La Roche). Total protein concentration was measured according to Lowry (Sigma kit). The AFP preparation was a homogeneous protein with a molecular weight of 70 kDa according to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and FPLC data (Severin et al., 1996). AFP-Dr conjugates synthesis AFP-Dr conjugates were obtained with watersoluble carbodiimide (EDAC: 1-ethyl-3-(3diethylaminopropyl)-carbodiimide; Sigma) as described previously (Severin et al., 1995). Dr was obtained from the Scientific Research Institute for Investigation of New Antibiotics of I.M.Sechenov Moscow Medical Academy. All the conjugates were prepared at 4C in 0.01  pyridine buffer, pH 5.0, containing a 2500-fold molar excess of EDAC relative to AFP. The conjugates were dialysed 3 times against PBS and their cytotoxic activity was examined. Protein concentration in the conjugates was assayed by the Lowry method. Dr concentration was measured as its optical density at 480 nm. The AFP–Dr molar ratio was 1:5.

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ovarian carcinoma: SKVLB which is Dr resistant and SKOV3 which is Dr sensitive. The resistant lines MCF-7 AdrR and SKVLB were obtained from the parental lines (MCF-7 and SKOV3) by selection for resistance to adriamycin and vinblastin (Bradley et al., 1989). The cell lines mentioned above were cultured in plastic flasks (Costar) in RPMI 1640 medium supplemented with 10% fetal calf serum (Gibco), 100 units/ml of penicillin and 100 ìg/ml of streptomycin (Gibco) in 5% CO2 at 37C. In order to maintain the resistance, SKVLB and MCF-7 AdrR cells were incubated once a month in a medium containing 0.4 ìg/ml of daunomycin for the selection and maintenance of the resistant population. Cytotoxicity of Dr and AFP-Dr conjugates The survival rate of cells under the action of various preparations was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test (Mosmann, 1983; Alley et al., 1988) by incubation in 96-well culture plates with different concentrations of the tested compound (in triplicate for each concentration) for 3 days; then 200 ìl of the culture medium were supplemented with 50 ìl of the MTT solution (1 mg/ml) for 2–4 h (Mosmann, 1983). The MTT stock solution was prepared as follows: MTT was dissolved in phosphatebuffered saline PBS (5 mg/ml), filtrated through a 0.45 ìm filter unit (Nalgene type SCN) and stored at 4C for a maximum of 1 month. The MTT solution in a culture medium was prepared immediately before the application. After staining the medium was removed the formazan crystals were dissolved in 150 ìl of dimethylsulfoxide (Alley et al., 1988), and the optical density was measured at 540 nm using a microculture plate reader Multiskan MS (Labsystems). Cell survival was estimated as a percentage of the corresponding control. Cytotoxicity of the drugs was assessed by the IC50, corresponding to the 50% decrease in cell survival rate.

RESULTS

Cell lines

Overcoming the resistance of SKVLB cell line by AFP-Dr conjugates

Two pairs of cell lines were used in this study. These are two cell lines of breast carcinoma: MCF-7 AdrR (Dr-resistant line) and MCF-7 (the initial line sensitive to Dr) and two cell lines of

It is well known that one of characteristic features of the multidrug resistance phenomenon based on hyperexpression of mdr1 is its reversal in vitro by verapamil inhibiting Pgp-mediated removal of the

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drugs from the cell (Sikic, 1993; Skovsgaard et al., 1994; Broxterman et al., 1995). Figure 1 shows that Dr-resistant cells of ovarian carcinoma line SKVLB were almost an order of magnitude more sensitive to the antibiotic in the presence of 20 ì of verapamil. The IC50 value for Dr was 7.5 ìg/ml and 1 ìg/ml, correspondingly. IC50 for the cells of the initially Dr-sensitive line SKOV3 was about 0.3 ìg/ml. Thus, this is a true example of the classic phenomenon of MDR. The data comparing the cytotoxic activity of free Dr and Dr-human AFP conjugates towards the Dr-resistant cell line SKVLB are presented in Figure 2. It is seen that in the case of 72-h drug incubation (Fig. 2(a)), as well as when the drug is removed 1 h after the beginning of incubation (Fig. 2(b)) the AFP-Dr conjugate significantly increased the sensitivity of highly resistant tumour cells. The sensitivity of antibiotic-resistant cells to Dr increased 10-fold when the drug was presented in the form of AFP-Dr conjugate, i.e. the IC50 value for Dr decreased, correspondingly, from 11 ìg/ml to 0.8 ìg/ml in the first case (Fig. 2(a), curves 1 and 2) and from 25 ìg/ml to 2.7 ìg/ml in the second case (Fig. 2(b), curves 1 and 2). By comparison, the IC50 value for Dr after reverting the resistance by verapamil was 1 ìg/ml (72-h incubation, Fig. 1). Thus, the AFP-Dr conjugate increases the sensitivity of the Dr-resistant cell line SKVLB (a human ovarian carcinoma) even more efficiently

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Fig. 1. Overcoming the multidrug resistance in human ovarian carcinoma by verapamil. The cell survival rate was evaluated 72 h after Dr addition to the Dr-resistant ovarian carcinoma cell line SKVLB (1); after Dr addition and 20 ì verapamil (2) to the SKVLB line; and after Dr addition to the initial antibiotic-sensitive human ovarian carcinoma cell line SKOV3 (3). (), IC50 =0.3 ìg/ml; (), IC50 =1.0 ìg/ml; ( ), 7.5 ìg/ml.

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Fig. 2. Overcoming the multidrug resistance in the human ovarian carcinoma cell line SKVLB by AFP–Dr conjugate. The cell survival rate was evaluated after incubation of the Dr-resistant ovarian carcinoma cell line SKVLB with free Dr (1) and with AFP–Dr conjugates (2). (a): cells were incubated with Dr and with AFP–Dr conjugate for 72 h; (b): cells were incubated with Dr and with AFP–Dr conjugate for 1 h, then the cells were washed-out. (), IC50 =0.8 ìg/ml; ( ), IC50 =11.0 ìg/ml; ( ), IC50 =2.7 ìg/ml; (), IC50 =25.0 ìg/ml.

than verapamil under the same experimental conditions. Overcoming the resistance of MCF-7 AdrR cell line by AFP-Dr conjugates Analogous experiments on the ability of AFP-Dr conjugates to decrease cellular resistance to drugs were conducted on the two cell lines of the human breast carcinoma: Dr-resistant cell line MCF-7 AdrR and the Dr-sensitive cell line MCF-7. As depicted in Figure 3, the Dr-resistant MCF-7 Adr line cells became nearly five times more sensitive to the antibiotic in the presence of 20 ì verapamil, i.e. the IC50 of Dr decreased from 7.3 ìg/ml to 1.6 ìg/ml. However, the Dr toxicity towards the parental Dr-sensitive cell line MCF-7 was two orders of magnitude higher than that

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Fig. 3. Overcoming the multidrug resistance in human breast carcinoma by verapamil. The cell survival rate was evaluated 72 h after Dr addition to the Dr-resistant breast carcinoma cell line MCF-7 AdrR (1); after Dr addition and 20 ì verapamil to the MCF-7 AdrR line (2); and after Dr addition to the initial antibiotic-sensitive human breast carcinoma cell line MCF-7 (3). ( ), IC50 =7.3 ìg/ml; ( ), IC50 =1.6 ìg/ml; ( ), 0.08 ml.

towards the corresponding resistant line as distinct from the SKVLB and SKOV3 lines. The IC50 for Dr towards the cell line MCF-7 was 0.08 ìg/ml (72-h incubation without washing-out). The AFP-Dr conjugates efficiently reverted MDR on the human breast carcinoma cell line as well (Fig. 4). In 72-h incubation experiments (Fig. 4(a)) as well as in experiments where the preparations were washed-out one hour after the beginning of incubation (Fig. 4(b)), AFP-Dr conjugates significantly increased the sensitivity of initially Dr-resistant breast carcinoma cells. In the first case, the IC50 value decreased from 7.2 ìg/ml to 2.0 ìg/ml, while in the second case it decreased from 56 ìg/ml to 13 ìg/ml. DISCUSSION The experimental results suggest that in contrast with the studied cell lines of ovarian carcinoma, in experiments whith human breast carcinoma neither the AFP-Dr conjugate nor verapamil did not fully overcome the MDR. We may suggest that the rationale for this phenomenon is in the existence of not one but several (at least two) mechanisms of cellular resistance. For example, there could be quantitative or qualitative changes in the activity of topoisomerase II or overexpression of other membrane proteins, such as MRP (MDR-associated protein), a rather frequent mechanism in cell lines obtained by selection for anthracyclin resistance (Skovsgaard et al., 1994; Chao, 1995; Versantvoort et al., 1995). In fact, cellular resistance is usually multifactorial.

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Fig. 4. Overcoming the multidrug resistance in breast carcinoma by AFP–Dr conjugate. The cell survival rate was evaluated after incubation of Dr-resistant breast carcinoma cell line MCF-7 AdrR with free Dr (1) and with AFP–Dr conjugates (2). (a): cells were incubated with Dr and with AFP–Dr conjugate for 72 h; (b): cells were incubated with Dr and with AFP–Dr conjugate for 1 h, then the cells were washed-out. ( ), IC50 =7.2 ìg/ml; (), 2.0 ìg/ml; (), 56 ìg/ml; ( ), 13 ìg/ml.

At the same time, the increase in the sensitivity of the Dr-resistant cells line MCF-7 AdrR in the presence of verapamil was approximately the same as that in the presence of AFP-Dr conjugates. Consequently, the cellular resistance component determined by hyperexpression of mdr1 is circumvented with equal efficiency by AFP-Dr conjugates in both studied lines (ovarian carcinoma and breast carcinoma). In summary, it may be concluded that using the oncofetal protein AFP as a vehicle for receptormediated transport of anticancer drugs allowed us to obtain not only highly selective and cytotoxic anticancer drugs, but also the preparations were simultaneously efficient towards human tumour cells that are highly resistant due to hyperexpression of the membrane protein Pgp. There exist recommendations (Veltishchev et al., 1991) for efficient chemotherapeutic treatment of

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