EFFECTS OF TAXOL ON THE HUMAN NCI-H295 ADRENOCORTICAL CARCINOMA CELL LINE
F. Fallo, C. Pilon, L. Barzon, M. Pistorello, U. Pagotto, G. Altavilla1, M. Boscaro and N. Sonino
Division of Endocrinology and 1Department of Pathology, University of Padova, Italy
ABSTRACT
We investigated the effects of taxol, an antimicrotubule agent active in different cancers, on the human NCI-H295 steroid-secreting adrenocortical carcinoma cell line. Cells were incubated for 48, 72 or 96 h with taxol 10-10-10-4 M. Cell viability was evaluated by MTT assay with IC50 calculation. Apoptosis was investigated by measuring DNA fragmentation with ELISA assay after cell exposure to taxol at IC50 for 24 h. For secretion studies, aldosterone, cortisol, testosterone and dehydroepiandrosterone-sulphate (DHEA-S) were measured by RIA in the conditioned medium after 96 h exposure to taxol 10-10-10-6 M, and expressed as percentage of steroid production by control cells. By MTT, taxol induced a dose- dependent inhibition of cell proliferation, with ICs50 at 72-96 h corresponding to blood levels achieved in vivo in patients with other types of cancer. Nuclear fragmentation, morphologically confirmed at electron microscopy, showed a 4-fold increase after exposure to taxol. With 10-6 M taxol, aldosterone decreased to 48%, cortisol to 61%, testosterone to 76% and DHEA-S to 89% of steroid production by control cells. Taxol is an effective cytotoxic and antiproliferative agent in a human adrenocortical steroid-secreting carcinoma cell line. Apoptosis induced by the drug is involved in neoplastic cell death.
INTRODUCTION
Adrenocortical carcinoma is a rare neoplasm 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, 2). Recently, the permanent culture of human NCI-H295 steroid-secreting adrenocortical carcinoma cells has become available to study the effects of antitumoral drugs either on cell proliferation or steroid production (3). Only suramin and mitotane have been proven to induce significant cytotoxicity in this cell line (4, 5). Taxol, a plant- derived antimicrotubule compound, is an effective cytoxic agent both in vitro and in vivo for cancers refractory to other drugs (6, 7). To examine the potential use of taxol for adrenocortical carcinoma, we assessed its effects on NCI-H295 cell line. Since taxol is able to activate the programmed process of cell death, i.e. apoptosis (8), we explored this phenomenon as a possible mechanism of interference with cell proliferation. The effect on steroid production by this cell line was also investigated.
MATERIALS AND METHODS
Materials
Taxol (as paclitaxel), purchased from Sigma Chemicals (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 tetrazolium bromide) was obtained from Sigma Chemicals.
Cell culture
The cell line NCI-H295 was obtained from American Type Culture Collection (USA). The cells (passages 60-70) were maintained in RPMI 1640 medium supplemented with 2% fetal calf serum, insulin (1 µg/ml), L-glutamine (2 mM), transferrin (1 µg/ml), selenium (1 ng/ml), and antibiotics (100 I.U./ml penicillin G Na, 100 µg/ml streptomycin sulfate, 250 µg/ml amphotericin B). Cells were maintained and grown on 75-cm2 flasks at 37℃ in humidified atmosphere of 5%
EFFECTS OF TAXOL
CO2 in air. During the initial three months of culture, only attached cells were retained when medium was changed. All experiments were performed on exponentially growing cells. For this purpose, NCI-H295 cells were plated in 24- 48 well tissue culture dishes at a density of 3-5 x 10+ cells in 0.5 ml/well for proliferation studies and of 2 x 105 cells in 0.5 ml/well for secretion studies, respectively, and allowed to attach and grow for 3 days. The seeding media were then changed with the experimental media. Cell doubling time was ~ 72 hours.
Cell proliferation studies
Incubations in the culture medium for 48 h, 72 h or 96 h at 37℃ with taxol concentrations ranging from 10-10 to 10-+ M were carried out in separate experiments. Cells were then washed twice with PBS, and cell viability was scored by MTT assay (9). The mitochondrial dye MTT was added to each well at 1 mg/ml and incubation was continued for 4 h. 0.04 M HCL in a SDS 20% solution was finally added. Absorbance was read at 550 nm on a Diagnostic Pasteur LP200 microplate reader (USA). Background absorbance was subtracted, and replicate wells were averaged. The 50% inhibitory concentration (IC50) value, i.e., drug concentration promoting the 50% loss of viable cells in comparison with control cells, was calculated.
Apoptosis was evaluated by determination of cytoplasmic histone-associated DNA fragments using an ELISA assay (Boehringer Mannheim, Germany), after exposure of cell line to taxol for 24 h in the conditioned medium at the IC50 calculated at 72 h. Results were expressed as absorbance read at 405 nm on a VmaxTM Kinetic microplate reader (USA). Background absorbance was subtracted, and replicate wells were averaged.
To examine apoptosis, in addition to routine staining for light microscopy, ultrastructural studies were performed. After washing, cells were fixed in a glutaraldehyde (4%) solution in PBS 0.2 M pH 7.2 at 4℃. The cells were then post-fixed in osmium tetroxide (1%), dehydrated and embedded in Epon 812, and observed using a Hitachi H7000 (Japan) transmission electron microscope.
Steroid determinations
NCI-H295 cells were exposed for 96 h to taxol 10-10-10-6 M. Aliquots (50- 100 ul) of the conditioned media were assayed for aldosterone, cortisol,
testosterone and dehydroepiandrosterone-sulphate (DHEA-S) by RIA, and compared with medium from control cells without taxol. Aldosterone was measured using antibodies kindly provided by Dr. S. Lewicka (10). Cortisol and testosterone were measured by commercial kits from Diagnostic Systems Labs (USA). DHEA-S was measured by a kit from BioRad Labs (USA).
Statistics
The results are expressed as mean + SEM. Triplicate determinations from duplicate experiments were considered for calculations. Group means were compared using analysis of variance followed by Fisher’s protected least significant difference test. P < 0.05 was considered significant.
RESULTS
Taxol induced a dose-dependent inhibition of cell proliferation as measured using MTT, with IC50 = 2 x 10-6 M at 48 h, IC50 = 3 x 10-7 M at 72 h and IC50 = 8 x 10-9 M at 96 h. The dose-response curve at 96 h is shown in Fig. 1. When NCI-H295 cells, treated for 96 h with taxol 10-6 M, were kept for an additional period without the drug, growth was not restored (data not shown).
In comparison with controls, DNA fragmentations showed a 4-fold increase after 24 h exposure to taxol. The phenomenon of apoptosis was morphologically confirmed by both light and electron microscopy evaluation. By the latter, cells showed chromatin aggregation as well as nuclear and cytoplasmic condensation into a cluster of membrane bound segments (apoptotic bodies), with preservation of morphologically intact mitochondria.
In the presence of taxol 10-10-10-6 M, a decreased production of aldosterone, cortisol, testosterone and DHEA-S was observed at 96 h in the cell line. At taxol 10-6 M, levels in comparison with untreated control cells were 84+3 vs 40±2.5 nmol/L/106 cells for aldosterone (P < 0.05), 393±25 vs 239±12 nmol/ L/106 cells for cortisol (P < 0.05), 27±1.4 vs 20±1.5 nmol/L/106 cells for testosterone (P < 0.05) and 1678±76 vs 1498±62 nmol/L/106 cells for DHEA-S (P < 0.05). Values expressed as percentage of steroid production by control cells are shown in Fig. 2.
100
Survival (% of control)
IC50 = 8 x 10-9 M
50
0
10-10
10-9
10-8
10-7
10-6
10-5
10-4
Taxol (M)
Viability of NCI-H295 cells after 96 h exposure to taxol at varying concentrations. The 50% inhibitory concentration (IC50) value is indicated. Means ± SEM of triplicate determinations from duplicate experiments are shown.
100
Hormone levels in colture medium (% of control)
Aldosterone
Cortisol
80
Testosterone
DHEA-S
60
40
20
0
FIGURE 2 Effect of 96 h exposure to taxol 10-6 M on steroid production by NCI-H295 cells in vitro. The results are expressed as percentage of steroid hormone production by an equal number of plated cells not exposed to taxol.
DISCUSSION
Our results show a relevant growth-inhibitory activity of taxol in a human model of adrenocortical steroid-secreting carcinoma cell line. The in vitro antiproliferative effect of taxol was pronounced at a range of concentrations comparable to those employed in other types of cancer cell line (6), and was increased over time. The effect appeared irreversible, since cell proliferation did not return to normal after withdrawal of the drug from the medium. The antiproliferative action of taxol has been related either to its direct cytotoxic property or to its ability to activate the programmed cell death process, i. e. apoptosis (11). This is confirmed in our experimental model, where cells exposed to taxol showed a marked increase of nuclear DNA fragmentation, which is a hallmark of apoptosis. Light microscopy and ultrastructural findings clearly displayed the morphological features typical of this phenomenon (12). Taxol ICS50 observed in our cell line model at 72-96 h were much lower than mean plasma concentrations achieved in vivo in patients after therapeutic intravenous administration for various types of cancer (13). All these data support a potential role of the drug in the treatment of patients with adrenal cancer in vivo, prospecting phase II clinical trials.
The effect on steroid hormone production from NCI-H295 cell line paralleled antitumor activity in vitro. In fact, 4-day exposure of cells to taxol 10-6 M resulted in a reduction of all adrenal steroids measured. A much weaker inhibitory effect on testosterone (76% of control cells) and DHEA-S (89%) than on aldosterone (48%) and cortisol (61%) production was observed. A preferential inhibition of some steroidogenic enzyme sites with an intracellular shift towards the formation of adrenal androgens over the time of cells exposure to taxol could explain these findings.
In conclusion, taxol is an effective cytotoxic and antiproliferative agent in a human adrenocortical steroid-secreting carcinoma cell line. Whether the drug may have antitumor activity in vivo in patients with adrenal cancer needs further investigation. A decrease in adrenal steroid production should be regarded as potential side-effect in patients given the drug for treatment other types of cancer.
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