Inhibition of proliferation by plasma concentrations of Car, Pac and both Car-Pac in NSCLC cells was investigated. In A-549 cells, Pac induced a time- and concentration-dependent inhibition of proliferation of 39.4%, 74.1% and 86.9% at 24 h, 48 h and 72 h, respectively. Low doses of Pac induced a minor rate of proliferation inhibition (27.4%, 58.1% and 77.7% at the same times). By contrast, high doses of Pac induced a similar rate of proliferation inhibition as the plasma concentrations (38.2%, 73.3% and 86.4%; see Figure 1A). Similar effects were observed in A-427 cells (Figure 1A). However, NODO cells showed a greater resistance to Pac activity. As shown in Figure 1A, Pac plasma concentrations induced only a 3.8%, 18.1% and 37.5% inhibition of proliferation at 24 h, 48 h and 72 h, respectively (Figure 1A).

Car plasma concentrations also induced a time- and concentration-dependent inhibition of proliferation, but this inhibition was much less than Pac. As shown in Figure 1B, A-549 and A-427 cells showed similar proliferation inhibition at 48 h (24.4% and 24.9%, respectively), while in NODO cells, this inhibition was 12.7%. At 72 h, all lung cancer cells showed similar proliferation inhibition (23.7% 22.5% and 25.4% in A-549, A-427 and NODO cells, respectively). Similar rates of proliferation inhibition were found with high and low doses of Car in A-549 and A-427 cells (Figure 1B). By contrast, high doses of Car at 72 h caused a high proliferation inhibition in NODO cells (41.7%; see Figure 1B).

The combination of plasma concentrations of Car and Pac had a cumulative effect at 24 h in A-549 and A-427 cells. The effect of these concentrations reached a plateau at 72 h by saturation of the mechanisms of cell death (Figure 2). By contrast, in NODO cells, the Car-Pac combination enhanced the proliferation inhibition (45.3%, 74% and 84.3% at 24 h, 48 h and 72 h, respectively) more than the sum of the effect of both drugs used independently (Figure 2).

Plasma concentrations of Pac induced apoptosis at rates of 25.3%, 53.3% and 51.3% in A-549 and at 15.4%, 30.2% and 56.6% in A-427 cells at the times analyzed (24 h, 48 h and 72 h). However, metastasis-derived NODO cells showed significantly lower rates of apoptosis (5.1%, 14.3% and 25.4% at the same times; see Figure 3A). Carboplatin at plasma concentrations induced a lower level of apoptosis than Pac in all cell lines, especially at the more prolonged exposure times (16.3% and 14.1% in A-549 cells and 12.8% and 17% in A-427, at 48 h and 72 h, respectively). The lower percentage of apoptosis was found in NODO cells. Car-Pac in combination produced a significant increase in percent apoptosis in all cell lines. Rates of apoptosis of 66.7%, 72.6% and 46.5% were reached in A-549, A-427 and NODO cells, respectively, at 72 h of drug exposure (Figure 3A). Apoptosis induction was confirmed using confocal laser-scanning microscopy (Figure 3B).

Expression of resistance genes was detected in NSCLC cell lines before and after drug treatment. A-549 cells showed MDR1 relative expression of 4.4 ± 0.3 after 8 h of Pac plasmatic concentration exposure, which increased to 7.5 ± 0.4 after 72 h after exposure (Figure 4A). NODO cells showed MDR1 relative expression of 6.9 ± 0.3 at 8 h and 7.4 ± 0.4 at 72 h. By contrast, A-427 cells showed relative expression of only 2.9 ± 0.1 at 8 h after exposure without significant changes at the different times analyzed. Carboplatin did not induce any modulation of MDR1 expression of the three lines (Figure 4A). Interestingly, combined Pac-Car treatment induced a greater MDR1 expression than the Pac exposure alone, which was particularly significant in A-549 (10 ± 0.2 at 8 h and 12 ± 0.8 at 72 h) and in A-427 cells (7.5 ± 0.3 at 8 h and 5.6 ± 0.3 at 72 h). NODO cells showed a low increase in the relative MDR1 expression with 3.5 ± 0.3 at 8 h and 6.1 ± 0.5 at 72 h after exposure (Figure 4A). By contrast, Pac did not modulate MRP3 expression, while Car induced a slight increase of MRP3 expression in A-549, A-427 and NODO cell lines. These modulations were maintained over time. Unlike the case of MDR1, the Pac-Car combination did not enhance MRP3 expression in NSCLC cell lines (Figure 4B).

The lung carcinoma A-549 (CCL185) and A-427 (HTB-53) cell lines were obtained from ATCC and grown with Ham’s F12K and DEMEM medium, respectively (Sigma Chemical Co., St. Louis, MO, USA), supplemented with 10% heat-inactivated foetal bovine serum (FBS), 40 mg/L gentamycin and 500 mg/L ampicillin (Antibioticos S.A., Spain). Cells were maintained in a monolayer culture at 37 °C in an atmosphere containing 5% CO₂. The NODO cell line (32050004) was developed from a lung cancer metastasis (Red de Bancos de Tumores, Granada, Spain) and was grown in an RPMI 1640 medium supplemented with 1% ITS (insulin-transferrin-selenium).

Cells were seeded in 24-well plates (10⁴ cells per well). MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] solution (5 mg/mL) was added to each well (20 μL) and incubated for 4 h at 37 °C. After the medium was removed, 200 μL of dimethylsulfoxide (DMSO) was added to each well. Optical density was determined using a Titertek multiscan colorimeter (Flow, Irvine, CA, USA) at 570 nm and 690 nm. The proliferation effect of Car, Pac and the Car-Pac combination was determined for 72 h. Plasma concentrations of Pac (5 μM) and Car (30 μM) [35,36], and higher and lower plasma concentrations of the drugs, were used for the assays. Morphological changes were analyzed by optical microscopy.

For analysis of the cell-cycle distribution, cells were harvested, washed twice with sample buffer (100 mg glucose; 100 mL PBS without Ca²⁺ or Mg²⁺), and fixed in 70% (v/v) cold ethanol for at least 1 h before staining. The cells were pelleted, washed once with sample buffer and resuspended in propidium iodide (PI) solution (50 μg/mL PI, 0.5 mg/mL RNase in sample buffer, pH 7.4) for 30 min in the dark. A fluorescence-activated cell sorter analysis was performed at 24 h, 48 h and 72 h after treatment. The data were collected and analyzed using the Cellfit program with a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). To confirm apoptosis, cells were washed twice with PBS and incubated in binding buffer containing annexin V-FITC (25 μg/mL) and PI (25 μg/mL) in the dark for 15 min at room temperature (Annexin V-FITC Apoptosis Detection Kit I; BD Pharmingen, San Diego, CA, USA). Alternatively, the fluorescence was detected by confocal microscopy using a Leica DMI6000 microscope (Heidelberg, Germany).

Total RNA was extracted from cells using the Rneasy Mini kit (Qiagen). cDNA was generated using the Promega Reverse Transcription System (Promega, Madrid, Spain) using total cellular RNA (1 μg). Polymerase chain reaction (PCR) amplification of the resistance genes was performed using primers specific to MDR1 [37] and MRP3 [10]. RNA integrity was assessed by amplification of β-actin mRNA [37]. PCR products were analyzed by standard agarose gel electrophoresis (data not shown). In order to quantify resistance gene expression, TaqMan quantitative real-time PCR was performed using cDNA corresponding to 40 ng RNA per reaction. Primers specific for MDR1, MRP3 and β-actin and TaqMan Fast Mastermix (Applied Biosystems) were used and amplifications carried out on the Applied Biosystem 7500 (Applied Biosystems, CA, USA). Gene expression levels were calculated by the ΔCt method: ΔCt = mean value Ct (mRNA reference) − mean value Ct (mRNA of interest). Normalised ΔCT (delta cycle threshold) values were obtained by subtracting the β-actin Ct from the gene of interest Ct. The relative expression of mRNA of the gene of interest corresponded to the 2^ΔCt value.

Statistical analyses were performed using the SPSS statistical software, version 16.0 (SPSS Inc., Chicago, IL, USA). The results were compared using the student’s t-test. All data are expressed as mean ± SD. Differences were considered statistically significant at a p value of less than 0.05.