Variable P-glycoprotein immunoreactivity unrelated to cytotoxic drug resistance in vitro of human adrenocortical carcinoma
CARIN BACKLIN1, HELENA FRIDBORG2, CLAES JUHLIN1, PETER NYGREN3, GÖRAN ÅKERSTRÖM1, JONAS RASTAD1 and ROLF LARSSON2
1Departments of Surgery; 2Clinical Pharmacology and 3Oncology, University Hospital, S-751 85 Uppsala, Sweden Received June 17, 1994; Accepted July 19, 1994
Abstract. Human adrenocortical tissue was immuno- histochemically investigated with monoclonal antibodies C219 and JSB-1 for expression of P-glycoprotein (Pgp) associated with resistance to multiple cytotoxic drugs. All normal (n=7) and adenomatous (n=3) glands strongly expressed the protein, while the cancers (n=15) demonstrated variable Pgp reactivity. Cytotoxic drug sensitivity testing in vitro on freshly dispersed cells from one normal adrenal gland and from 6 carcinomas with no to extensive Pgp reactivity demonstrated universally poor sensitivity to 9 standard drugs including those with Pgp mediated resistance. The findings indicate that immunohistochemical staining for Pgp cannot be used to predict cytotoxic drug resistance and that other mechanisms mediate cytotoxic drug resistance in adrenocortical carcinoma.
Introduction
A transmembrane phosphoglycoprotein (Pgp) of 130-180 kDa has been associated with multiple cytotoxic drug resistance in a variety of malignant neoplasms (1) and possibly mediates extrusion of cytotoxic drugs across the tumor cell membrane (2). This resistance may be modulated with several substances like verapamil, diltazem and cyclosporin A in vitro by possibly competing with cellular mechanisms mediating cytotoxic drug extrusion (3,4).
Patients with adrenocortical carcinoma have dismal prognosis and cure virtually depends on possibilities for radical surgery alone (5-7). This tumor rarely responds to chemotherapy in vivo, although transient regression has been reported notably with mitotane (o,p’DDD) alone (8,9) or in combination regimens (10,11). Unequivocal expression of Pgp and its mRNA has been suggested by analysis on limited samples of adrenocortical carcinoma (1,12-15), while
cytotoxic drug resistance of these neoplasms in vitro and its relationship to Pgp await clarification.
This study investigates immunohistochemical Pgp expression in normal and neoplastic human adrenal cortex in relation to cytotoxic drug sensitivity in vitro, whereby it is substantiated that other mechanisms than Pgp possibly mediates the meagre response of adrenocortical carcinomas to cytotoxic regimens in vivo.
Materials and methods
Patients and tissues. Seven microscopically normal human adrenal glands were obtained in connection with nephrectomy for renal cell carcinoma. Histologically benign adrenocortical adenomas were excised from 3 patients. These lesions exhibited a greatest diameter of 1.5-3.5 cm, and they were associated with cortisol, aldosterone or no measurable hormone excess upon analysis of blood and urine (16). Cortical carcinomas were acquired during routine adrenalectomy of 13 patients and another 2 contributed with local recurrencies after such operations. The primary cancers displayed tumor cell invasion into vessels (n=5) or the peripheral capsule (n=4) in combination with unequivocal but variably extensive parenchymal cell atypia, nuclear pleomorphism, mitoses, and necroses. The longest diameter of these lesions exceeded 10 cm (mean, 15.6 cm), while the recurrences measured 6 and 12 cm. Preoperative screening of basal steroid hormones in blood and urine revealed excess cortisol (n=3), aldosterone (n=2), androgens (n=5), and estrogen (n=3) either alone or in different combinations, while 6 cancers were considered as overtly non- hyperfunctioning (Table I). All but one of the patients with adrenal malignancy demonstrated the pathological urinary steroid profile of cortical carcinoma (17) with increased levels of mainly 3ß-hydroxy-5-ene steroids and tetrahydro- 11-deoxycortisol. Among the patients subjected to primary adrenal surgery, 2 individuals had metastases at the time of operation and another 5 developed biochemical or radiological signs of tumor recurrence during postoperative follow-up for mean 2.9 years. All patients with adrenal adenomas are biochemically cured upon reinvestigation 3-5.3 years postoperatively. Patients providing normal or neoplastic adrenal specimens revealed history of no antitumor treatment prior to surgery.
Correspondence to: Dr Jonas Rastad, Department of Surgery, University Hospital, S-751 85 Uppsala, Sweden
Key words: human adrenocortical carcinoma, P-glycoprotein, cytotoxic drug resistance, immunohistochemistry, fluorometry, dispersed cells
| Tissue | Steroid excess | Parenchymal reactivity | |
|---|---|---|---|
| extentª | intensityb | ||
| normal | none | >50/>50 | ++/++ |
| AA 1 | cortisol | >50/>50 | ++/++ |
| AA 2 | aldosterone | >50/>50 | ++/++ |
| AA 3 | none | >50/>50 | ++/++ |
| AC 1 | cortisol, estrogen | >50/>50 | +/+ |
| AC 2 | cortisol, aldosterone | >50/>50 | ++/+ |
| AC 3 | cortisol, androgen | >50/0 | ++/- |
| AC 4 | aldosterone | >50/0 | ++/- |
| AC 5 | androgen | >50/<10 | ++ |
| AC 6 | androgen | >50/<10 | ++/+ |
| AC 7 | androgen | <10/<10 | +/+ |
| AC 8 | androgen, estrogen | <10/<10 | +/+ |
| AC 9 | estrogen | <10/>50 | +/++ |
| AC 10 | none | 0/<10 | -/+ |
| AC 11 | none | 0/0 | -/- |
| AC 12 | none | <10/>50 | +/+ |
| AC 13 | none | >50/>50 | ++/++ |
| AC 14 | none | <10/0 | +/- |
| AC 15 | none | >50/>50 | +/+ |
“demonstrates extent of parenchymal immunostaining as 01, <10%, 10-50%, >50%; bdemonstrates intensity of parenchymal reactivity arbitrily defined as -, +, ++.
Immunohistochemistry. The murine monoclonal IgG2a antibody C219 (Centocor Diagnostics, Malvern, PA) and the IgG1 antibody JSB-1 (Monosan, Sanbio, Am Uden, The Netherlands) were utilized for demonstration of Pgp (18,19). Tissue for immunohistochemistry was snap frozen at -70℃ in liquid isopentane and stored at this temperature until cryosectioning. Acetone-fixed sections, 6 um thick, were immersed into 0.3% H2O2 in phosphate buffered saline (PBS) for 15 minutes and exposed to normal rabbit serum diluted 1/10. The C219 and JSB-1 antibodies were serially diluted, whereafter dilutions of 1/6 and 1/20 were selected for investigations, respectively. The antibodies were applied for 30 minutes and at room temperature in PBS containing 1% bovine serum albumin (BSA; Sigma Chemicals, St Louis, MO). A rabbit anti-mouse antibody (Dako, Copenhagen, Denmark) diluted 1/40 was then applied for 30 minutes followed by mouse peroxidase-antiperoxidase complex (Dako) diluted 1/250 and ethylcarbazole in dimethyl- sulfoxide (DMSO; Sigma).
The normal human adrenal cortex was utilized as positive control in all stainings of the carcinomas. Omission of the primary antibody or application of the irrelevant IgG1 antibody E11 (10 µg/ml) were used as negative controls and yielded no immunostaining of the normal or neoplastic
adrenal specimens. The E11 antibody recognizes a large glycoprotein present on parathyroid, placental and renal tubule cells but not the human adrenal gland (20,21). All immunoreacted sections were evaluated blindly by 2 independent observers and re-evaluated together upon inconsistent findings. Presence of immunostaining was arbitrarily classified as +, ++ and as <10%, 10-50%, >50% according to the general intensity and the extent of parenchymal reactivities, respectively.
Cell preparation. Cells used in the cytotoxic assay were prepared as previously described (22). Briefly the normal and neoplastic cortical tissues were dissected and cut fine with scissors in cold Ham’s F10 culture medium (Flow, Irvine, Scotland). The tissue pieces were shaken with repeated pipette aspirations for 1 hour at 37℃ in the medium containing collagenase (Boehringer Mannheim, Mannheim, Germany; 2 mg/ml), DNAse (Sigma; 0.05 mg/ml), 1 mM Ca2+ and 1.5% BSA. After filtering through a nylon mesh of 75 um pore size, the suspensions were purified on 25% isotonic Percoll (Kabi-Pharmacia, Uppsala, Sweden) in PBS. Cell viability determined by Trypan blue exclusion routinely exceeded 95%.
Cytospin preparations of all cell suspensions intended for drug sensitivity testing in vitro were fixed in pure acetone and stained with the C219 antibody as described above. Parallel preparations were reacted with Mayer’s hematoxylin and with 4 recently generated monoclonal anti-adrenocortical antibodies (10 µg/ml). These murine antibodies react with majority of the parenchymal cells of the normal human adrenal cortex and together also with all hitherto investigated adrenocortical carcinomas (unpublished data). The latter stainings together with cytologic inspection of cytospins were utilized to microscopically substantiate tumor cell fractions exceeding 75% among samples intended for cytotoxic drug sensitivity testing in vitro.
Reagents and drugs for cytotoxicity assay. Fluorescein diacetate (FDA; Sigma) was dissolved in DMSO and kept at -20℃ as a stock solution (10 mg/ml) protected from light. The sources, handling and concentration ranges of investigated cytotoxic drugs have recently been described (23). In addition, karboplatin (Bristol-Myers Squibb, Bromma, Sweden) and the active metabolite 4-hydroperoxy- cyclophosphamide (4HC; Asta Pharma, Frankfurt, Germany) were diluted in PBS and analysed at cut-off concentrations (23) of 25 µg/ml and 2 ug/ml, respectively. Streptozocin (Upjohn, Partille, Sweden) and o,p’DDD (Serva, Heidelberg, Germany) were diluted in PBS and a mixture of ethanol and PBS, respectively.
Microtiter plates (Nunc, Roskilde, Denmark) for drug sensitivity testing were prepared by adding 20 ul/well of each drug solution at 10x the desired final concentration with a programmable pipetting robot (Propette; Perkin Elmer, Norwalk, CT) as described previously (23). The plates were stored at -70°℃ without apparent change in drug activity during 2 months. Final maximal solvent concentrations of 0.1% had no effect on cell survival.
Cytotoxicity assay. The principal steps of the fluorescent microculture cytotoxicity assay (FMCA) have been described
previously (24). After suspending the single and small clusters of adrenal cells at 5x104 per ml RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Flow), 2 mM glutamine, 50 µg/ml streptomycin and 60 ug/ml penicillin, 180 ul of this suspension was seeded into each well of the prepared plates. Six blank wells received the culture medium alone, while 6 wells with cells but no drugs served as control. Cells and media were added to the wells with the Propette robot. The culture plates were incubated with continuous drug exposure for 72 hours at 37℃ in humidified atmosphere containing 5% CO2. Thereafter the plates were centrifuged (200 g, 7 min) and the medium was removed by flicking the plate. Following a wash with PBS, 100 ul PBS containing 10 µg/ml FDA was added columnwise to control, experimental and blank wells with an automated 96-well dispenser (Multidrop; Flow). The plates were incubated for 1 hour before fluorescence recording in Fluoroscan 2 (Flow) blanked against wells containing FDA in PBS alone.
The scanning fluorometer Fluoroscan 2 is equipped with a xenon lamp and broad band interference filters providing excitation at 485 nm for FDA. Emitted light from a vertical light path of each well is sequentially read at 538 nm, which occupies approximately 1 min for each plate. The fluorescence data was transferred to a custom made software for automated dose-response analysis using Microsoft Excel (Microsoft, Berks, England), Apple Macintosh SE/30, and Kaleida graph (Synergy Software, Reading, PA).
Quantification of FMCA results. Only successful assays were evaluated and this required a flourescence signal of control cultures exceeding mean blank values by at least 5 times, mean coefficients of variation below 30% for the signal intensity of control cultures, and a proportion of tumor cells of at least 75%. The results are obtained as survival index (SI) defined as fluorescence in per cent of control cultures with blank values subtracted. Cytotoxic sensitivity data are presented as dose-response curves for indicated concentration ranges or as SI values at the drug cut-off concentrations defined previously (23). Briefly the cut-off concentration represents the level where each drug has presented maximal scatter of SI values upon analysis of more than 400 tumor samples from 10 different solid and hematological malignancies. Moreover this concentration has been found to predict drug sensitivity patterns similar to clinical phase II trials (25). Resistance profiles of peripheral leukocytes from 5 patients with untreated acute myeloblastic leukemia and dispersed cells of 4 patients with untreated non-Hodgkin lymphoma are included for comparison with fairly drug sensitive tumors.
Results
Immunohistochemistry. Immunoreactions of the C219 and JSB-1 antibodies appeared as a finely granular and dispersed precipitate, which mainly seemed to be confined to the cell membrane but also appeared in the cytoplasm and along the nuclear membrane of cells. All 7 normal adrenal glands showed distinct immunostaining with the antibodies on virtually all parenchymal cells of the cortex (Fig. 1), although
the reactivity was somewhat less intense in the zona glomerulosa. The generally sparse stroma of these glands expressed weak immunoreaction with both antibodies. The medulla remained unstained apart from the presence of intense reactivity confined to islands of cortical cells surrounded by the medullary parenchyma. All cortical adenomas demonstrated similarly intense and extensive Pgp reactivity as in the normal cortex (Table I).
The antibody reactivities generally were weaker and more heterogeneously distributed in the adrenocortical carcinomas as compared with the normal and adenomatous cortex (Fig. 2). Analysis with the C219 antibody substantiated absence to <10% parenchymal reactivity within 7 cancers, and that the others displayed >50% staining (Table I). Only 3 carcinomas demonstrated the immunoreaction on virtually all discernible parenchymal cells, while scattered cell groups of variable size were stained in the remaining immunoreactive cancers. Intense staining occurred in 5 tumors and merely encompassed those with more extensively distributed reactivity. Investigation with JSB-1 displayed a similarly dichotomous extent of Pgp expression. Below 10% reactive parenchyma was observed in 9 cancers, while the staining encompassed variable majorities in the others. The JSB-1 reactivity was considered intense in 2 extensively stained neoplasms. The stroma was unequivocally stained with the JSB-1 and C219 antibodies in 4 and 2 of the cancers, respectively, and all but one of them demonstrated generally limited parenchymal antibody reactivities.
Seven cancers (47%) demonstrated largely similar extent of staining with both antibodies, and encompassed 3 lesions with absence or <10% parenchymal reactivity (Table I). The remaining cancers showed discrepant extent of tumor cell reactivity and included 3 lesions displaying meager staining with one antibody combined with essentially complete labelling with the other. The staining extensiveness with C219 exceeded that of JSB-1 in all but 3 of the lesions with discrepant reactivity. The local carcinoma recurrencies (AC 1,8) revealed variable Pgp stainings. There was no obvious relationship between the presence or absence of parenchymal staining with either antibody and the pattern of peripheral steroid excess, tumor size, or clinical signs of local recurrence and metastases.
Cytotoxic drug sensitivity. The cryosectioned parenchyma of adrenocortical carcinomas analysed for drug sensitivity in vitro was unreactive (AC 11) or weakly to moderately stained with both Pgp antibodies (AC 5), and extensively stained by either C219 (AC 3) or JSB-1 (AC 9) or both (AC 1,13; Table I). The general extent of reactivity with C219 was confirmed upon analysis of cytospins from aliquots of cells subsequently investigated for drug sensitivity (not shown).
Among 9 commonly used cytotoxic drugs, the normal adrenocortical cells showed a resistant profile but for 4HC (Fig. 3A). The carcinoma preparations tended to display an even greater resistance to the investigated drugs (Fig. 3B-D), and this lack of response did not vary with the immuno- histochemical signs of Pgp expression. In contrast cells from patients with untreated non-Hodgkin lymphoma (Fig. 3E) and acute myeloblastic leukemia (Fig. 3F) showed more
A
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Melphalan
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5-fluorouracil
5-fluorouracil
Melphalan
Melphalan
Carboplatin
Carboplatin
Cisplatin
Cisplatin
Cyclophosphamide*
Cyclophosphamide*
Mitoxantrone
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op-DDD
op-DDD
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5-fluorouracil
Melphalan
Melphalan
Carboplatin
Carboplatin
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Cisplatin
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Cyclophosphamide*
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sensitive profiles especially for drugs known to be active in these diseases, e.g., mitoxantrone, etoposide, and doxorubicin. Only the normal cortical cells responded to high concentrations of streptozocin and, in comparison with the cancer cells, they were also more sensitive to increasing concentrations of o,p’DDD (Fig. 4).
Discussion
Adrenocortical carcinomas constitute rare tumors with overall 5-year survival rates generally ranging between 16% to 35% and approaching 47% upon macroscopically radical
resection (7,26,27). Several cytotoxic therapies including mainly cisplatin and doxorubicin have been attempted in vivo, but o,p’DDD alone or in combination regimens seems to be the most frequently administered drug (9,11). Interestingly o,p’DDD recently has been suggested to increase cytotoxic drug accumulation in a Pgp expressing cell line of human adrenocortical cancer (14). Effects of chemotherapy in human adrenocortical carcinoma nevertheless seem limited to essentially temporary partial responses. These findings coincide with the current demonstration of considerable resistance to conventional cytotoxic drugs within all aliquots of freshly dispersed cells from these neoplasms.
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Analysis of Pgp encoding RNA sequences has demonstrated considerable Pgp expression in normal human adrenal glands (12,14,28). Extensive and homogeneously intense Pgp immunoreactivity of the normal adrenal cortex was supported by the current analysis with the C219 and JSB-1 antibodies, which recognize multiple isoforms of highly conserved internal epitopes of Pgp (18,19). Hybridization as well as immunohistochemistry with these and other Pgp reactive antibodies have also substantiated Pgp expression in human adrenocortical carcinomas, but the levels have been variable and essentially analysed in limited tumor series (1,12-14). In a recent evaluation on the presence or absence of Pgp staining, however, 11 adrenocortical carcinomas all expressed epitopes recognised by a panel of Pgp reactive antisera (15), and this encompassed also the JSB-1 antibody despite its reduced reactivity with formalin- fixed specimens (19). These findings generally concur with the absence of parenchymal reactivity with the C219 and JSB-1 antibodies in merely a single of 15 primary and recurrent adrenocortical carcinomas.
Variable Pgp immunoreactivity was demonstrated by a majority of the currently investigated adrenocortical carcinomas. This observation related to discrepancies between antibody stainings and common limitation of parenchymal reactivities to cell groups of variable size. These findings may indicate failure to unveil modest Pgp expression sufficient for drug resistance. Immunohisto- chemical staining of RPMI 8226 myeloma cells with the C219 and JSB-1 antibodies, however, clearly visualized stepwise increments in Pgp expression between cells being sensitive and approximately 6-fold or 40-fold doxorubicin resistant (not shown). These observations support that the utilized technique of immunostaining can reveal Pgp expression associated with low to moderate drug resistance. It is possible that discrepancies between Pgp mRNA and protein expressions in adrenocortical cancers may relate to disturbances in Pgp synthesis and translocation to the tumor cell surface. Moreover, it should be emphasized that careful inspection is necessary to disclose Pgp confined to stromal tumor components alone (29). Recognized difficulties in
histopathological discrimination between benign and malignant adrenocortical neoplasms may also contribute to misinterpretations on the extent of cancer Pgp expression, since the currently investigated adenomas consistently reacted with the intense and widespread immunoreactivity of the normal human adrenal cortex.
Pgp constitutes a major mechanism contributing to multidrug resistance of tumor cells in vitro and in vivo, and the extent of Pgp expression has been correlated with the degree of drug sensitivity in vitro (4,30,31). These findings contrast to cells of the normal and malignant adrenal cortex, which showed essentially similar drug resistance profiles despite highly variable extent and intensity of Pgp immunoreactivity. It must be ascertained, however, that lack of cytotoxic responsiveness is unrelated to inadequate concentration or instability of investigated drugs. The currently utilized FMCA has been standardized with respect to drug exposure and stability (23,24), and the concomitantly investigated lymphoma and leukemia preparations displayed considerably greater drug sensitivity. This finding concurs with clinical experience of these diseases, and previous FMCA data have predicted chemotherapeutic responses in patients with acute leukemias (23). Taken together the immunohistochemical analysis and cytotoxic drug unresponsiveness of freshly prepared cells from human adrenal cortex indicate that other mechanisms than Pgp seem responsible for multidrug resistance of adrenocortical carcinomas. This conclusion is substantiated also by observations on meager effects of modulators of Pgp related drug resistance in human adrenocortical carcinoma (32).
Various clinically utilized substances can circumvent multidrug resistance through interference with Pgp mediated drug extrusion (4,33). Immunostaining for Pgp has been suggested as marker for clinical applicability of such therapeutic strategies (31). In view of the currently demonstrated discrepancies between Pgp expression and drug sensitivity within adrenocortical carcinoma, general optimization of chemotherapy on the mere basis of Pgp immunoreactivity seems doubtful. Resistance modulators, however, may potentiate cytotoxic drugs even in the absence
of Pgp expression, which indicates interaction also with other mechanisms mediating drug resistance (34). In absence of reliable markers for the benefit of resistance modulators, such as the previously proposed staining for Pgp, drug sensitivity testing in vitro is advocated, whereby net effects of all resistance mechanisms may be appreciated prior to chemotherapeutic modulation.
Acknowledgements
Dr Manuel de la Torre, Department of Pathology, is gratefully acknowledged for cytologic evaluation of cell preparations. This study was supported by the Swedish Cancer Society and the Swedish Medical Association.
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