We used a novel real-time method to analyze the cellular growth of the prostate cancer cell line LNCaP under different conditions. This method allowed us to determine the optimal time points for adding substances that affect cell growth, and reduced the likelihood of missing transient effects. We also characterized the effect of phenol red and fetal bovine serum on LNCaP cell growth, and their interference with hormone stimulation and bicalutamide (Casodex) treatment.
LNCaP is a commonly used prostate cancer cell line derived from a lymph node metastasis; it is described as androgen sensitive . To investigate the influence of additives on cell growth, LNCaP must be cultured in phenol red–free media containing hormone-reduced fetal bovine serum (FBS) [2, 3]. Several studies have shown that LNCaP cells cease androgen-dependent growth behavior upon passage in phenol red–containing media [4, 5], and that phenol red promotes cell growth in a physiologically abnormal manner . To analyze cell growth in real time, we used the xCELLigence System, which measures the electrical impedance during cell growth on gold electrodes . We were thus able to determine the influence of media components on LNCaP cell growth without stimulation, after stimulation with 5a-dihydrotestosterone (DHT), and after addition of bicalutamide (Casodex), an androgen receptor antagonist known to affect LNCaP cells .
Results and Discussion
In our first experiment, we compared the growth of 1 x 104 LNCaP cells/well (96-well MTP) in RPMI (plus phenol red) supplemented with 10% FBS (= normal medium) with growth in RPMI (no phenol red) with 10% hormone-reduced FBS (reduced medium). In both media, we treated the cells with DHT (50 nM) or bicalutamide (5 µM or 20 µM). There was a weak, 1.3-fold increase in cell growth in normal media in the presence of DHT, but growth was reduced in the presence of bicalutamide. We also observed that while DHT did not reduce cell adhesion within the first 8 hours, cell adhesion was reduced by bicalutamide (Figure 1a and 1b). In reduced medium, the stimulatory effect was seen 8–21 hours after seeding of 1 x 104 cells/well (96-well MTP). At that time, DHT stimulation resulted in a twofold increase in cell growth (Figure 1c and 1d). The reduction in cell growth after addition of 20 µM bicalutamide was comparable in the two media (Figure 1c). Simultaneous addition of DHT and bicalutamide resulted in reduced growth compared with untreated cells, and initial stimulation of cell growth by DHT was blocked by later addition of bicalutamide (data not shown).
We next added a higher DHT concentration of 100 nM and analyzed LNCaP growth in RPMI without phenol red but supplemented with normal FBS. In this experiment, we were able to discriminate between the effects of phenol red and FBS. We found that the effects of DHT and bicalutamide on cell growth were greatest 24–48 hours after seeding of 2 x 104 cells/well (96-well MTP). Within this time range, cell growth in medium without phenol red but with normal FBS was far more weakly stimulated compared with cells grown in medium without phenol red but with hormone-reduced FBS; stimulation was completely abolished in growth medium with phenol red (Figure 2a). We also observed that the inhibitory effect of bicalutamide on cell growth was present only up to 42 hours after its addition. Experiments with lower bicalutamide concentrations revealed that the reduction in cell growth was greatest and most dose dependent when the cells were grown in RPMI containing phenol red and normal FBS. If phenol red was omitted, a significant reduction in cell growth was observed after 10 µM bicalutamide was added, whereas for cells grown in medium without phenol red and with hormone-reduced FBS, no significant reduction in cell growth was seen with either 5 µM or 10 µM bicalutamide (Figure 2b).
Our results suggest that the influence of androgen receptor–stimulating or –blocking substances strongly depends upon the composition of the growth medium. The use of hormone-depleted FBS in combination with phenol red–free medium is strongly recommended for monitoring stimulator effects, whereas inhibitory effects may be more readily analyzed in medium containing normal FBS. Because the mutated androgen receptor in LNCaP can also be stimulated by estrogens, progestogens, and several antiandrogens , we suggest that the stimulatory effect of normal FBS compared with hormone-reduced FBS may be explained by differences in estradiol levels (192 pg/ml in normal FBS compared with <12 pg/ml in hormone-reduced FBS) rather than by differences in progesterone levels (0.29 ng/ml in normal FBS compared with 0.13 ng/ml in hormone-reduced FBS) or testosterone levels (0.08 ng/ml in normal FBS compared with 0.03 ng/ml in hormone-reduced FBS). Our results show that the optimal time point for monitoring inhibitory and stimulatory effects on cell proliferation depends strongly upon cell number; therefore, real-time cell analysis is a perfect tool for detecting these effects.
This work was supported by grants from the Deutsche Krebshilfe and the Stiftung Europrofession.
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