Chemotherapy 1998;44:129-134
Francesco Falloª Catia Pilona Luisa Barzona Matteo Pistorelloa Uberto Pagottoª Giuseppe Altavillab Marco Boscaroa Nicoletta Soninoa
a Division of Endocrinology, Institute of Semeiotica Medica, University of Padova, and
b Department of Pathology, University of Padova, Italy
Paclitaxel Is an Effective Antiproliferative Agent on the Human NCI-H295 Adrenocortical Carcinoma Cell Line
Abstract
In view of a potential clinical use, we assessed the antiprolifer- ative effect of paclitaxel on the human steroid-secreting NCI- H295 adrenocarcinoma cell line. By MTT, paclitaxel induced a dose-dependent inhibition of cell proliferation, with IC50 lower than blood levels of the drug achieved in patients treated for other malignancies. Cell exposure to paclitaxel for 24 h at the different IC50s produced a dose-responsive increase in DNA fragmentation, morphologically confirmed by electron microscopy. A time-dependent decrease in aldosterone, corti- sol and testosterone was observed. Paclitaxel is an effective antiproliferative agent in this human adrenocortical carcino- ma cell line. Apoptosis induced by the drug is involved in neo- plastic cell death. A potential role of the drug in the treatment of patients with adrenocortical cancer could be considered.
Key Words
Adrenal cancer Paclitaxel Apoptosis Steroids NCI-H295
Introduction
Adrenocortical carcinoma is a rare neo- plasm and is often diagnosed at an advanced stage, so that therapeutic intervention proves of very little benefit. The use of adrenalytic or conventional chemotherapeutic agents has yielded modest results, and their clinical use is limited by toxicity [1-3]. Paclitaxel (formu- lated as Taxol®), a plant-derived compound with a novel cytotoxic mechanism of action, is
an effective agent both in vitro and in vivo for cancers refractory to other drugs [4, 5]. By shifting the cellular equilibrium toward mi- crotubule assembly, paclitaxel causes the for- mation of disorganized microtubule bundles that interfere with normal cellular function during interphase and mitosis, resulting in cell death [6, 7]. Microtubules are involved in the steroidogenic response of either normal or tumor adrenocortical cells to corticotropic factors [8].
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To examine the in vitro antineoplastic ac- tivity of paclitaxel in view of a potential clini- cal use, we assessed its effects on the human steroid-secreting NCI-H295 adrenocarcino- ma cell line recently characterized [9, 10]. Since paclitaxel is able to activate the pro- grammed process of cell death, i.e. apoptosis [11, 12], we explored this phenomenon as a possible mechanism of interference with cell proliferation. The effect on steroid produc- tion by this cell line was also investigated.
Materials and Methods
Materials
Paclitaxel, purchased from Sigma (St. Louis, Mo., USA), was dissolved in dimethylsulfoxide and then diluted into cell growth medium for use in assays. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetra- zolium bromide) was also obtained from Sigma.
Cell Culture
The cell line NCI-H295 was obtained from the American Type Culture Collection (Rockville, Md., USA). The cell (passages 60-70) were maintained in RPMI 1640 medium supplemented with 2% fetal calf serum, insulin (1 µg/ml), L-glutamine (2 mM), trans- ferrin (1 µg/ml), selenium (1 ng/ml) and antibiotics (100 IU/ml penicillin G Na, 100 µg/ml streptomycin sulfate, 250 µg/ml amphotericin B). Cells were main- tained and grown on 75-cm2 flasks at 37℃ in humidi- fied atmosphere of 5% CO2 in air. During the initial 6 months of culture, only attached cells were retained when the medium was changed. All experiments were performed on exponentially growing cells, routinely maintained as monolayer cultures. To this purpose, NCI-H295 cells were plated in 24- to 48-well tissue cul- ture dishes at a density of 3-5 × 104 cells in 0.5 ml/ well for proliferation studies and of 2 × 105 cells in 0.5/ml well for secretion studies, and allowed to attach and grow for 3 days. The seeding media were then changed with the experimental media. Cell doubling time was ~ 72 h.
Cell Proliferation Studies
Incubations in the culture medium for 24, 48, 72 and 96 h at 37°℃ with Taxol concentrations ranging from 10-10 to 10-4 M were carried out in separate experiments. Cells were then washed twice with PBS,
and cell viability was scored by MTT assay [13]. The mitochondrial dye MTT was added to each well at 1 mg/ml, and the incubation was continued for 4 h. Hydrochloride 0.04 M in an SDS 20% solution was finally added. Absorbance was read at 550 nm on a Diagnostic Pasteur LP200 microplate reader (Austria). Background absorbance read at 690 nm was sub- tracted, and replicate wells were averaged. The 50% inhibitors concentration (IC50) value, i.e. drug concen- tration promoting the 50% loss of viable cells in com- parison with control cells, was calculated.
Quantitative measurement of apoptosis was car- ried out with the Cell Death Detection enzyme-linked immunosorbent assay kit from Boehringer Mannheim (Mannheim, Germany), following the manufacturer’s instructions. This method is based on the photometric immunoassay of cytoplasmic histone-associated DNA fragments. In the same experiment, cells (5 × 104 in 0.5 ml/well) were exposed to paclitaxel for 24 h in the conditioned culture medium at the IC50 calculated at 24, 48, 72 and 96 h. Results were given as enrichment factors, i.e. the specific enrichment of mono- and oligo- nucleosomes released by treated cells, calculated as absorbance of treated cells/absorbance of control cells. Absorbance was read at 405 nm on a VmaxTM Kinetic microplate reader (Medgenix, Belgium). Background absorbance read at 490 nm was subtracted, and repli- cate wells were averaged.
To examine apoptosis, in addition to routine stain- ing for light microscopy, ultrastructural studies were performed. After washing, cells were fixed in a glutar- aldehyde (4%) solution in PBS 0.2 M (pH 7.2) at 4°C. The cells were then postfixed in osmium tetroxide (1%), dehydrated and embedded in Epon 812, and observed using a Hitachi H7000 (Japan) transmission electron microscope.
Steroid Determination
NCI-H295 cells were exposed for 24, 48, 72 and 96 h to paclitaxel 10-6 M. Aliquots (50-100 ul) of the conditioned media were assayed for aldosterone, corti- sol and testosterone by radioimmunoassay, and com- pared with medium from control cells without pacli- taxel. Cell number and viability were determined at the beginning and at the end of experimental periods by the trypan blue exclusion method. Aldosterone was measured using antibodies kindly provided by Dr. S. Lewicka [14]. The detection limit was 0.05 nmol/l; the intra- and interassay coefficient of variations (CVs) were 8.4 and 11.8%, respectively. Cortisol and testos- terone were measured by commercial kits from Diag- nostic Systems Labs (Webster, Tex., USA). The limit of detection for cortisol was 3 nmol/l; the intra- and
Fallo/Pilon/Barzon/Pistorello/Pagotto/ Altavilla/Boscaro/Sonino
interassay CVs were 6.9 and 9.1%, respectively. The limit of detection for testosterone was 0.28 nmol/l; the intra- and interassay CVs were 8.5 and 10.7%, respec- tively.
Statistics
Results are expressed as means ± SE. Triplicate determinations from triplicate experiments were con- sidered for calculations. Statistics were performed us- ing StatView (Abacus Concepts, USA) on a Macintosh computer. Group means were compared using analysis of variance followed by Fisher’s protected least signifi- cant difference test. A p < 0.05 was considered statisti- cally significant.
Results
Paclitaxel induced a dose-dependent inhi- bition of cell proliferation as measured using MTT, with IC50 = 2 x 10-6 M at 24 h, IC50 = 5 x 10-7 M at 48 h, IC50 = 6 x 10-8 M at 72 h and IC50 = 6 x 10-9 M at 96 h. The dose- response curves at subsequent time points are shown in figure 1. When NCI-H295 cells, treated for 96 h with paclitaxel 10-6 M, were kept for an additional period without the drug, growth was not restored (data not shown).
Cell exposure to paclitaxel for 24 h at the different IC50s produced a dose-responsive in- crease in DNA fragmentation (enrichment factors 4.6 ± 0.2, 5.8 ± 0.2, 7,2 ± 0.4, 9.6 ± 0.4 with IC50 calculated at 96, 72, 48, 24 h, respectively). The phenomenon of apoptosis was morphologically confirmed by both light- and electron-microscopic evaluation. By the latter, cells showed chromatin aggregation as well as nuclear and cytoplasmic condensa- tion into a cluster of membrane-bound seg- ments, i.e. apoptotic bodies, with preserva- tion of morphologically intact mitochondria (fig. 2a, b).
As shown in table 1, in comparison with untreated control cells, cells treated with pa- clitaxel 10-6 M showed a time-dependent de-
100
24 h
48 h
72 h
96 h
Survival (% of control)
50
0
10-10
10-9
10-8
10-7
10-6
10-5
10-4
Paclitaxel (M)
crease in aldosterone, cortisol and testoster- one in culture medium. Values expressed as percentage of steroid production by control cells are shown in figure 3.
Discussion
The human adrenocortical carcinoma has been found to be highly resistant in vitro, by different mechanisms, to several anticancer drugs [15, 16]. Since the steroid-secreting NCI-H295 adrenocarcinoma cell line has be- come available to study the effects of adrena- lytic agents either on proliferation or steroid production, only suramin and mitotane have been proven to possess a significant cytotoxic acitivity in these cells. However, NCI-H295 cells are resistant to concentrations of either
A
2a
2b
100
Aldosterone
Hormone levels in culture medium (% of control)
& Cortisol
Testosterone
80
60
40
20
0
24
48
72
Time (h)
96
3
| Time h | Aldosterone | Cortisol | Testosterone | |||
|---|---|---|---|---|---|---|
| control | paclitaxel | control | paclitaxel | control | paclitaxel | |
| 24 | 13.4±0.7 | 7.5±0.4* | 161.9±4.2 | 135.1±3.7* | 4.4±0.1 | 4.2±0.2 |
| 48 | 19.2±1.0 | 9.4±0.5* | 309.1±8.7 | 217.3±5.5* | 11.6±0.6 | 10.1±0.4* |
| 72 | 32.6±2.0 | 15.1±1.0* | 428.2±11.3 | 231.8±6.3* | 13.9±0.3 | 11.3±0.3* |
| 96 | 59.1±2.3 | 14.2±0.6* | 534.1±17.4 | 161.1±5.9* | 19.1±0.9 | 14.3±0.7* |
The values (means ± SE of three separate experiments in triplicate) represent the steroid concentration in the conditioned culture medium after 24-96 h exposure to paclitaxel 10-6 M.
* p <0.05 vs. untreated control cells.
Fallo/Pilon/Barzon/Pistorello/Pagotto/ Altavilla/Boscaro/Sonino
suramin or mitotane lower than those clinical- ly achievable for therapeutic purposes with- out excessive toxicity [17-19].
Our results show a time-dependent and rel- evant antiproliferative activity of paclitaxel in this human cell line model at a range of con- centrations comparable to those employed in other types of cancer cell line [20-22]. A direct comparison of paclitaxel with other an- titumor agents has not been formally done in our study. However, the IC50S at 72-96 h appeared 400- to 4,000-fold lower than that reported for mitotane after 7 days of exposure in the same type of cell line [19]. The effect of paclitaxel was irreversible, since cell prolifera- tion did not return to normal after withdrawal of the drug from the medium. Moreover, pa- clitaxel IC50s at 72-96 h were markedly lower than mean plasma concentrations achieved in vivo in patients after therapeutic intravenous administration for various types of cancer [23]. The antiproliferative action of paclitaxel has been related either to its direct cytotoxic property and/or to its ability to activate the apoptotic process [7, 11, 12]. This is con- firmed in our experimental model, where cells exposed to paclitaxel showed a marked in- crease in nuclear DNA fragmentation, which is a hallmark of apoptosis [24]. Light-micro- scopic and ultrastructural findings clearly dis- played the morphological features typical of this phenomenon [25]. Cell cycle inhibitors p21 and p53 [26] as well as major histocom- patibility complex class II [27] have been recently shown to be involved in the mecha- nisms leading to apoptosis in normal human adrenocortical cells, and could also be in- volved in paclitaxel-induced apoptosis of the NCI-H295 cells.
The effect of steroid hormone production from the NCI-H295 cell line paralleled antitu- mor activity in vitro. In fact, exposure of cells to paclitaxel at a dose very close to IC50 at 72 h resulted in a time-dependent reduction
in all adrenal steroids measured. This per- centage inhibition of cell proliferation was similar to the percentage reduction in aldoste- rone and cortisol production at the same time of exposure to paclitaxel. This would suggest that in vitro the inhibitory effect of steroid production by paclitaxel was due to the sup- pression of cell growth per se. On the other hand, a much weaker inhibitory effect on tes- tosterone was observed at all time points. A preferential inhbition of some steroidogenic enzyme sites with an intracellular shift to- wards the formation of adrenal androgens by paclitaxel could explain these findings. In this respect, a direct effect of paclitaxel on steroid biosynthesis in the adrenocortical cells has been previously suggested [28]. A decrease in adrenal steroid production should be re- garded as a potential side effect in patients given the drug for treatment of other types of cancer.
In conclusion, paclitaxel is an effective an- tiproliferative agent in a human adrenocorti- cal steroid-secreting carcinoma cell line. Al- though caution is advised for the extrapola- tion of our in vitro results to in vivo condi- tions, a potential role of the drug in the treat- ment of patients with adrenocortical cancer could be considered.
Acknowledgments
This work was supported by grant 667/01/96 from ‘Regione Veneto, Giunta Regionale-Ricerca Sanitara Finalizzata’, Venezia, Italy.
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