NSCLC cell lines, A549 (ATCC^® CRL-185™) and H2170 (ATCC^® CRL-5928™) were cultured in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) medium containing 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 µg/ml streptomycin (all purchased from ATCC) and grown at 37°C in a humidified 5% CO₂ atmosphere. Human lung fibroblast (IMR-90) cells were cultured in MEM-α (1x)-Glutamax medium containing 10% FBS, 100 IU/ml penicillin and 100 µg/ml streptomycin (ATCC). Cells were maintained in T75 tissue culture flasks, and the medium was changed three times a week. Confluent cells were harvested by washing in phosphate-buffered saline (PBS) followed by trypsinisation (0.25% in EDTA) for subculturing. All of the cell lines were purchased from ATCC, and culture reagents were purchased from Gibco-Life Technologies (Grand Island, NY, USA) unless otherwise stated.

The NSCLC cell lines (A549 and H2170) were harvested upon incubation with 0.25% trypsin (Life Technologies, Foster City, CA, USA) and washed with phosphate-buffered solution with 2% FBS. The CD166-PE and EpCAM-FITC (BD Biosciences, San Jose, CA, USA) antibodies were used for CSC identification by flow cytometry. Briefly, cells were trypsinised, counted by a haemocytometer and transferred to 75-mm polystyrene round-bottom test tubes (BD Falcon, NJ, USA) at a cell concentration of 1×10⁶ cells/ml and subsequently stained with 10 µl of antibodies in the dark at 4°C. The cells were then washed and filtered through a 40-µm cell strainer to obtain a single-cell suspension before sorting. The expression of cancer stem cell markers (CD166 and EpCAM) was analysed and sorted using FACSAria III (BD, Biosciences). Gating used for the sorting of CD166⁺/EpCAM⁺ (Q2) and CD166⁻/EpCAM⁻ (Q3) NSCLC cell lines is depicted in Fig. 3.

Sorted lung tumour cells (1.0×10³ cells/ml) were suspended in serum-free medium containing DMEM F12 (Gibco) supplemented with 10 ng/ml fibroblast growth factor (bFGF), 1% of B27, 20 ng/ml of EGF, 1% antibiotic-antimycotic, (all purchased from Life Technologies) and seeded in an 96-well ultra-low attachment (ULA) dish. Spheroid formation was assessed by light microscopy after 20 days of culture. Self-renewal capabilities were also evaluated by monitoring single cells using the live cell analyser (JuLI™ Br; NanoEnTek, CA, USA).

Curcumin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 1 ml DMSO to make a stock solution of 10 mM. The curcumin stock was then diluted in complete RPMI-1640 medium to provide a substock and final working concentrations. Cisplatin (Sigma-Aldrich) was prepared as a 10 mM stock in 0.9% sodium chloride (NaCl) and was diluted in complete RPMI-1640 medium to provide a substock. The solution was filtered through a 0.22-µm membrane, aliquoted and stored at −20°C until further use.

IC₅₀ values for the single treatment with either curcumin and cisplatin of NSCLC cell lines were assessed by the MTS [3-(4,5-dimethylthiazol-2-yl)-2H-tetrazolium, inner salt] assay purchased from Promega (Madison, WI, USA). Tumour cells were plated at a density of 1×10⁴ cells/well in 96-well plates and incubated overnight in humidified air with 5% CO₂ at 37°C. NSCLC cells were then treated with a working concentration of curcumin (10, 20, 30 and 40 µM) and cisplatin (5, 10, 15, 20 and 25 µM) for 48 h. After a 48-h incubation, 15 µl of MTS solution was added to each well and incubated for another 4 h. Solubilisation solution (100 µl) was later added to the cells, and the absorbance at 570 nm was measured using Odyssey^® SA Imaging System (Li-Cor, Lincoln, NE, USA), using wells without cells as the blank. Cell viability was calculated according to the following formula: Cell viability (%) = cells (sample)/cells (control) × 100 and IC₅₀ was calculated using log formula.

In order to evaluate the efficacy of curcumin sensitisation prior to cisplatin treatment in NSCLC cell lines, both A549 and H2170 cells were initially sensitised/cultured with different doses of curcumin (10, 20, 30 and 40 µM) for 24 h, followed by low dose cisplatin (<3 µM) for another 24 h. Briefly, the tumour cells were seeded (1.0×10⁵ cells/well) in 6-well plates and sensitised with different doses of curcumin for 24 h. On the following day, the cells were harvested and seeded again in 96-well plates (1.0×10⁴ cells/well) with medium containing cisplatin (low dose) for another 24 h. At the end of the experiment, 15 µl of MTS solution was added to each well and incubated for another 4 h. Solubilisation solution (100 µl) was later added to the cells, and the absorbance at 570 nm was measured using Odyssey SA Imaging System, using wells without cells as the blank.

The IC₅₀ values of both curcumin and cisplatin in the A549 and H2170 cells were tested on IMR-90 cells to evaluate the toxic effect of curcumin and cisplatin on normal cells. IMR-90 cells were seeded overnight in 96-well plates at a density of 1.0×10⁴ cells/well in 100 µl complete MEM-α. Subsequently, 100 µl of either curcumin and/or cisplatin (concentration based on IC₅₀ of A549 and H2170) was added to the cells and incubated for 48 h. The viability of the IMR-90 cells was assessed by adding 10 µl of Presto Blue (BD Pharmingen, Franklin Lakes, NJ, USA) to each well and incubated for 2 h before the absorbance was measured at 570 nm.

The apoptosis assay was conducted using the Annexin V/propidium iodide (PI) apoptosis kit purchased from BD Pharmingen. In brief, 9.0×10⁵ cells/well of sorted and unsorted NSCLC cells were seeded into 6-well plates and incubated overnight. Direct combination (synergistic effects) of both curcumin and cisplatin on the NSCLC cell lines was performed by incubation of the cells in medium containing the single treatment (cisplatin or curcumin) and combination of both using the IC₅₀ doses for 48 h. Indirect combination (sensitising effects) of curcumin was performed by incubating the NSCLC cell lines with curcumin (IC₅₀ value) for 24 h, followed by incubation with low dose cisplatin (3 µM) for another 24 h. After treatments for 48 h (synergistic and sensitisation), both NSCLC cell lines were harvested by trysinisation and collected by centrifugation. The cell pellet was suspended in 100 µl of 1X Annexin V binding buffer (Becton Dickinson BD) and 1 µl of Annexin V-FITC was added. Antibody incubation was performed at 4°C for 20 min, and 1 µl of PI was later added before FACS acquisition. Stained cells were subjected to flow cytometric analysis using a FACSCalibur instrument (Becton Dickinson BD), and a total of 10,000 events were acquired and analyzed using Cell Quest software (Becton Dickinson BD).

Briefly, sorted and unsorted NSCLC cells were seeded at a density of ~3–4×10⁵ cells/well in complete medium and grown overnight to a 90% confluent monolayer. The next day, the cells were treated with colcemide (10 µg/ml) for 2 h for cell synchronisation. After incubation, a scratch wound was inflicted using a sterile 200-µl pipette tip and gentle washing was carried out twice using PBS to remove debris. Cells were then incubated with 2 ml of media containing both single treatments (cisplatin and curcumin) and/or the combination of both for another 48 h. The concentration of curcumin and cisplatin (single treatments and/or combination) used for the assay was based on the IC₅₀ values evaluated on both A549 and H2170 cells. Images of migrated cells (five fields of each triplicate well) were captured using relief contrast microscopy at ×40 magnification (Olympus IX 71; Olympus, Tokyo, Japan) and analysed for 48 h. The numbers of cells that migrated into the wound area were evaluated using the formula: Percentage of migrated cells = [initial scratch (0 h) − final scratch (48 h)]/initial (0 h) × 100.

NSCLC cells (A549 and H2170) were seeded in 6-well plates at a density of 9.0×10⁵ cells/well and incubated overnight. After incubation, the cells were treated with IC₅₀ values of curcumin and cisplatin, respectively for 48 h. Cells were then harvested and washed with ice-cold PBS. Antibodies (CD326 and CD166) (10 µl) were added to each tube and incubated for another 20 min in the dark at 4°C. The cells were then suspended in ice-cold PBS supplemented with 2% FBS, and a total of 10,000 events were acquired and analysed using Cell Quest software (Becton Dickinson BD).

Initially, total RNA was extracted and evaluated for purity as previously described. Transcriptor First Strand cDNA synthesis kit (Roche Applied Science, Nonnenwald, Penzberg, Germany) was used to synthesise cDNA according to the protocols recommended by the manufacturer. Quantitative RT-PCR (qRT-PCR) was performed using the Light Cycler 480 (Roche, Mannheim, Germany), on sorted lung tumour cells subsequent to treatment either by single agent (cisplatin or curcumin) or direct combination of both (synergistic effects) based on the IC₅₀ values. The qRT-PCR reaction was prepared using SYBR 1 Master Mix (Roche Applied Science, Penzberg, Germany) and primers as stated in Table I. PCR conditions were set under the following cycle conditions: pre-denaturation for 4 min at 95°C followed by 40 cycles consisting of denaturation at 95°C for 15 sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec followed by dissociation curve. The basic relative gene expression (RQ) was calculated using the ΔΔCt formula and the efficiency (E) of primer binding equal to 2.

All data are expressed as the mean ± standard deviation (SD) of three independent experiments. Comparison between two groups was performed using the two-tailed t-test. P-values of <0.01 were considered to indicate statistically significant differences.

The IC₅₀ values of A549 and H2170 cells treated with curcumin and cisplatin were assessed by MTS assay at 48 h. The results showed that exposure of NSCLC cell lines (A549 and H2170) to a range of curcumin (≤40 µM) and cisplatin (≤25 µM) concentrations resulted in IC₅₀ values of 41 and 30 µM and 33 and 7 µM, respectively (Fig. 1A and B). Furthermore, we noted that the IC₅₀ values for both A549 and H2170 cells to curcumin were almost equal. However, the IC₅₀ value of cisplatin in the H2170 cells was markedly lower compared to that for the A549 cells indicating higher sensitivity of H2170 cells to cisplatin-induced inhibition. Based on the IC₅₀ value indicated, the combination of 41 µM curcumin and 30 µM cisplatin was selected for A549 cells and 33 µM of curcumin and 7 µM of cisplatin were selected for H2170 cells for further downstream study (Table II).

To determine whether curcumin sensitises NSCLC cell lines to the tumour inhibitory effect of low dose cisplatin (≤3 µM), NSCLC cells (A549 and H2170) were incubated overnight with different doses of curcumin (10, 20, 30 and 40 µM), harvested and seeded again with low dose cisplatin (≤3 µM) for another 24 h. Treated NSCLC cells were subsequently evaluated for cell viability using the MTS assay. Treatment of the A549 and H2170 cells with curcumin (10–40 µM) markedly enhanced the sensitivity of both NSCLC cell lines to cisplatin (Fig. 2A and B).

Treatment of both A549 and H2170 cells with 3 µM cisplatin alone (low dose) was found to be ineffective to induce inhibition of growth in the NSCLC cell lines (tumour viability ~80%). However, this treatment became highly effective similar to the IC₅₀ concentration (tumour viability ~50%) when both A549 and H2170 were initially sensitised to different concentrations of curcumin (10–40 µM) (Fig. 2C and D). Moreover, exposure of IMR-90 cells to the IC₅₀ values of curcumin and cisplatin in both NSCLC cell lines did not induce toxicity to the cells, as the percentage of viability was higher than 90% (Fig. 2E).

The expression of CSC markers (CD166 and EpCAM) in the NSCLC cell lines was studied and we found a small population of NSCLC cells (A549 and H2170) that showed positivity for CD166 and EpCAM (CD166⁺/EpCAM⁺) (Fig. 3). The NSCLC cells showed consistent double-positive expression of CD166⁺/EpCAM⁺ ranging from 3.0 to 4.5% (Fig. 3). This was consistent with the characteristics of CSCs in a tumour population indicating that the expression of CSC markers should be within ~4% of the total population (56). Moreover, the double-negative (CD166⁻/EpCAM⁻) population was much lower in the NSCLC cell lines with 1.7 and 0.3% in both the A549 and H2170 cells, respectively (Fig. 3A and B). The CD166⁺/EpCAM⁺ and CD166⁻/EpCAM⁻ populations were sorted from the A549 and H2170 cells into a 15-ml tube containing complete medium and transferred to a T75 flask for expansion and further downstream study.

The ability of the double-positive (CD166⁺/EpCAM⁺) subpopulation sorted from the NSCLC cell lines to form three-dimensional spheres in serum-free medium containing stem cell growth factors (EGF and bFGF) on non-adherent plates was examined. The sorted NSCLC cells grew as anchorage-independent spheres after 14 days of culture (Fig. 4B). We observed that the isolated CD166⁺/EpCAM⁺ subpopulations of both A549 and H2170 cells were able to form tumour spheres with an average size ranging from 50 to 200 µm in diameter (Fig. 4B). We also noted that the CD166⁺/EpCAM⁺ subpopulation isolated from A549 cells had the ability for self-renewal and produced daughter cells, which is an important characteristic of CSCs (Fig. 5B). However, there were cells observed as non-dividing (Fig. 5A), which were a dormant phenotype of CSCs.

In order to study the combination effects of curcumin and cisplatin on the regulation of CSC subpopulations, the expression of two combination markers (CD166 and EpCAM) which were previously described as markers of lung cancer CSCs, were evaluated in the NSCLC cell lines (A549 and H2170) after treatment with either a single agent of curcumin or cisplatin, or a combination of both. Treatment of A549 and H2170 cells with the combination of curcumin and cisplatin led to an average increase of ~10 and ~6% expression of the double-positive (CD166⁺/EpCAM⁺) CSC subpopulation, respectively, as compared to both curcumin and cisplatin treatment alone (Table III). Combination treatment only led to ~2% increase and a slight reduction (~0.5%) in the CD166⁻/EpCAM⁻ subpopulation noted in the A549 and H2170 cells, correspondingly (Table III), suggesting that the combination of both curcumin and cisplatin synergistically acted to enrich the double-positive (CD166⁺/EpCAM⁺) subpopulation as compared to each single treatment alone.

The apoptotic effects of the combined treatment of curcumin either by synergism with cisplatin or sensitising effects prior to treatment with low dose cisplatin in the double-positive (CD166⁺/EpCAM⁺) CSC subpopulation was examined using the apoptosis assay (Annexin V/PI) 48 h post-treatment. The unsorted A549 and H2170 cells were used as a control to indicate the basal level of apoptosis following the treatments (Fig. 6). As shown for the synergistic effect of curcumin (Fig. 6A); the results indicated that single treatments of curcumin and cisplatin induced apoptosis in the double-positive (CD166⁺/EpCAM⁺) CSC subpopulation of A549 cells to an average of 14 and 15% following 48 h of treatments. Moreover, the apoptotic effect was significantly increased to an average of ~37% in the double-positive (CD166⁺/EpCAM⁺) CSC subpopulation as these two treatments were applied simultaneously. The results also showed that curcumin sensitisation prior to treatment with low dose cisplatin in the double-positive (CD166⁺/EpCAM⁺) CSC subpopulation of A549 cells substantially increased the percentage of apoptosis by ~20% as compared to treatment with low dose cisplatin with only 2% apoptosis (Fig. 6A).

There were no significant changes in the percentage of apoptosis between the single treatment of curcumin or cisplatin, and the combination treatments by synergistic effects on sorted H2170 cells (Fig. 6B). This result suggests that the cells are highly sensitive to both curcumin and cisplatin; the IC₅₀ concentrations of both treatments given to the cells have already induced a maximal response. Interestingly, we noted that by sensitising the double-positive (CD166⁺/EpCAM⁺) CSC subpopulation of H2170 cells to curcumin, prior to treatment with low dose cisplatin notably enhanced its apoptotic effect by 40%, compared to only 20% apoptosis as observed for the synergistic treatments. Combination treatments by sensitisation of the double-positive CD166⁺/EpCAM⁺ CSC subpopulation of H2170 cells to curcumin, also significantly increased the percentage of late apoptosis to 35%, compared to treatment with low dose cisplatin with only 4% detected (Fig. 6B).

To evaluate the migratory potential of the CSC subpopulation in the NSCLC cell lines (A549 and H2170) and the effects of curcumin either alone or in combination with cisplatin to inhibit the migration of these cells; scratch wound (migration) assay was performed in the sorted (CD166⁺/EpCAM⁺ and CD166⁻/EpCAM⁻) and unsorted NSCLC cell lines 48 h post-treatment. As depicted in Fig. 7B and D, the double-positive (CD166⁺/EpCAM⁺) CSC subpopulation had a significantly higher migratory potential as compared to the unsorted cells observed for both NSCLC cell lines. The combination of curcumin and cisplatin reduced the percentage of cell migration from 33.8 (cisplatin) to 17.3% (combined) in the unsorted A549 cells (Fig. 7B) with no apparent changes as noted in the H2170 cells (Fig. 7D). Furthermore, combined treatment markedly inhibited the migration of the CD166⁺/EpCAM⁺ subpopulation from 19.6 to 8.7% in the A549 cells and from 32.6 to 20.9% in the H2170 cells as compared to cisplatin treatment alone (Fig. 7B and D). Moreover, curcumin alone was able to inhibit the migration of the CD166⁺/EpCAM⁺ subpopulation in both the A549 and H2170 cells signifying the potential of curcumin on CSC inhibition. A higher cell migration was also noted in the CD166⁻/EpCAM⁻ compared to the CD166⁺/EpCAM⁺ subpopulation for the cisplatin treatment alone in A549 cells, while an opposite effect was noted in the H2170 cells (Fig. 7B and D). However, there were no differences in the percentage of cell migration between the CD166⁺/EpCAM⁺ and CD166⁻/EpCAM⁻ subpopulation for the combination treatment in both NSCLC cell lines (Fig. 7B and D).

Finally, in order to understand the mechanisms behind the process, we investigated specific genes involved in apoptosis (Apaf1, cytochrome c and caspase-9) and cell cycle regulation (cyclin D1 and p21) in the double-positive (CD166⁺/EpCAM⁺) CSC subpopulation of both A549 and H2170 cells, after induction of treatments using either curcumin or cisplatin, and the combination of both. The results showed that the relative gene expression level of Apaf1 was higher in the combined treatment group compared to the single treatments (curcumin or cisplatin) in the CD166⁺/EpCAM⁺ subpopulation of A549 cells (Fig. 8A). Furthermore, the expression of p21 was high, with low expression of the cyclin D1 gene, in the CD166⁺/EpCAM⁺ subpopulation of both the A549 and H2170 cells, as compared to the CD166⁻/EpCAM⁻ subpopulation in the combined treatment group (Fig. 8A and B). Combined treatments induced high expression of caspase-9 in the CD166⁺/EpCAM⁺ subpopulation of A549, compared to single treatments of curcumin (Fig. 8A). On the other hand, the expression of caspase-9 was consistently low in the CD166⁺/EpCAM⁺ subpopulation of H2170 cells for all of the treatments (Fig. 8B).