Introduction
Although advances have been made in decreasing the mortality rates, the prognosis of lung cancer remains poor. With an overall 5-year survival rate of <18%, the poor prognosis of lung cancer is mainly due to lack of understanding of the mechanisms involved in oncogenesis and drug resistance to chemotherapy (1). Recent findings suggest that cancer stem cells (CSCs) may be responsible for tumorigenesis and contribute to resistance to chemotherapy (2,3). In those studies, it was found that tumors are organized in hierarchical heterogeneous cell populations, which constitute a spectrum of phenotypically different cell types. Among these cell subsets, CSCs have the ability to sustain tumor growth in a very small fraction of tumor cells and possess the characteristics of longevity, self-renewal, multilineage differentiation capacity and tumorigenicity. In addition, CSCs express high levels of the adenosine-triphosphate-binding cassette (ABC) transporter that is responsible for pumping out chemotherapeutic drugs (4). Thus, identification of CSCs may be useful to understand the mechanism of generation of lung cancer and identify more effective therapeutic methods.
A useful method for the isolation of CSCs is dependent on their ability to discharge lipophilic and fluorescent dyes such as Hoechst 33342 by the ABC membrane transporter. Based on the capacity to efflux Hoechst 33342 dye, side population (SP) cells are considered to be enriched for CSCs in most types of human tumors, such as glioma (5), pancreatic cancer (6), renal cell carcinoma (7), breast cancer (8), and nasopharyngeal carcinoma (9). Moreover, accumulating evidence shows that SP cells were described to possess cancer stem cell-like properties and may be a useful tool to identify cancer stem cell populations (10,11). However, to the best of our knowledge, few studies have examined the role of SP cells in lung cancer.
Autophagy is a highly conserved catabolic process among all eukaryotes involving the degradation of cellular organelles and proteins. In a previous study, we found that autophagy acted as a potential survival mechanism when the cancer cells were treated with chemotherapy, and inhibition of autophagy enhanced the lethal effect of cisplatin (12). In addition, recent evidence has indicated that several types of tumor are dependent on autophagy for growth under normal conditions (13). However, the exact role of autophagy in the survival of SP cells of lung cancer to chemotherapy has yet to be determined.
In the present study, we isolated SP cells from an A549 lung cancer cell line using a fluorescence-activated cell sorter (FACS). The cancer stem cell-like properties of SP cells were verified through confocal fluorescence imaging, sphere formation and cell proliferation and colony formation assays, and gene expression in vitro and tumor formation in vivo. We also investigated whether autophagy is involved in the survival of SP cells in lung cancer cell lines to cisplatin.
Materials and methods
Cell culture
A549 lung cancer cells were obtained from the Key Laboratory of The First Affiliated Hospital of Zhengzhou University, and were cultured in RPMI-1640 supplemented with 100 ml/l fetal bovine serum (FBS) and 100 U/ml penicillin/streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2.
FACS analysis and purification of SP cells
A549 cells were eluted with 2.5 g/l trypsin and 0.5 g/l ethylenediaminetetraacetic acid, centrifuged, washed and resuspended at 1×106 cells/ml in pre-warmed RPMi-1640 containing 2% FBS. The cells were then incubated for 120 min at 37°C with 5 µg/ml Hoechst 33342 alone or in combination with 50 µg/ml verapamil which inhibited ABC transporters. After the incubation process, the cells were washed in phosphate-buffered saline (PBS), centrifuged at 4°C and resuspended in PBS solution supplemented with 2% FBS and 1 mM HEPES. The cells were filtered through a 40 µm mesh filter and reserved at 4°C for flow cytometry analysis. The SP cells were selected and sorted by FACS (BD Biosciences, San Jose, CA, USA). The Hoechst dye was excited with a UV laser at 346 nm and its fluorescence was measured with 630/22 (Hoechst 33342 Red) and 424/44 filters (Hoechst Blue).
Confocal fluorescence imaging
A549 cells were washed, resuspended and incubated with 5 µg/ml Hoechst 33342 in the dark at 37°C for 30 min. The cells were washed again in PBS three times, and 400 µl PBS was added. Confocal microscopy was used to capture the images of SP cells.
Sphere formation assays
SP cells were seeded at a density of 1×102 cells/well in 6-well plates. The cells were cultured in Dulbecco’s modified eagle’s medium (DMEM)/F12 medium containing 10 mg/l insulin, 20 µg/l epidermal growth factor (EGF) and 10 µg/l basic fibroblast growth factor (bFGF). Insulin, EGF and bFGF were added every 3 days. After culture for 10–14 days, the formation of floating spheres was visually assayed. The spheres containing >30 cells were selected, and then trypsinized to gain the single cells. After suspension of these cells, the cells were added into 6-well plates at a density of 1×102 cells/well. The secondary sphere formation was assayed after culture for 10–14 days. The number of spheres was counted under the dissecting microscope.
Cell proliferation assay
SP and non-SP cells were counted and added into 96-well plates at a density of 104 cells/well. The cells were cultured in RPMI-1640 supplemented with 100 ml/l FBS for 5 days. A Cell Counting kit-8 (CCK-8) analysis was used to detect and compare the growth of these cells each day. The growth curve was plotted according to the absorbance of each well.
Colony formation assay
SP and non-SP cells were cultured in RPMi-1640 supplemented with 100 ml/l FBS at a density of 1×102 cells/well for 10–14 days. The number of clones that contained >50 cells were counted and recorded.
Gene expression
Total RNA was extracted from SP and non-SP cells separately using TRIzol reagent according to the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using the SuperScrit First-Strand Synthesis system (Invitrogen, Carlsbad, CA, USA) as described in instructions. The RT-PCR was carried out using the SuperScript One-Step kit (Invitrogen). The primers used were: MDR1, 5′-CCCATCATTGCAATAGCAGG-3′ and 5′-GTTCAAACTTCTGCTCCTGA-5′ for a 157-bp fragment; ABCG2, 5′-CTGAGATCCTGAGCCTTTGG-3′ and 5′-TGCCCATCACAACATCATCT-3′ for a 380-bp fragment; OCT4, 5′-CTGTAACCGGCGCCAGAA-3′ and 5′-TGCATGGGAGAGCCCAGA-3′ for a 218-bp fragment; GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′ for a 249-bp fragment. The PCR products were separated by electrophoresis in 2% agarose gel.
Tumor formation
SP and non-SP cells were sorted and resuspended at a density ranging from 104 to 103 cells. The cells were mixed with 50 µl Matrigel to prevent cell dispersion and loss. The cells were subcutaneously injected into 6- to 7-week-old nude mice, obtained from the Experimental Animal Center of Zhengzhou University, China. The mice were monitored to assess tumor formation for 8 weeks. The experiments were approved by the Laboratory Animal Ethics Committee of Zhengzhou University.
Autophagy expression
Cells were stained with 0.05 mol/l of monodansylcadaverine (MDC) solution for 15 min and observed using a confocal laser microscope to detect the autophagolysosomes. The cells were collected and lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor. Lysated proteins were centrifuged at 14,000 rpm for 10 min and quantified. Proteins were separated by a 4–20% gradient SDS/PAGE gel and transferred to polyvinylidene fluoride (PVDF) membranes. The membrane was placed in TBST solution containing 5% non-fat skim milk at room temperature for 1 h. The membranes were incubated with diluted primary antibodies overnight at 4°C and washed with TBST three times for 10 min. Subsequently, the membrane was reacted with secondary antibodies in 2.5% non-fat skim milk for 1 h at room temperature, followed by washing with TBST three times for 10 min. Blots were stripped and re-blotted with anti-β-actin, and the membrane was examined for the band.
Apoptosis assay
SP cells were treated with the chemotherapeutic drug cisplatin for 24 h and then incubated with or without 3-methyladenine (3-MA), which is an inhibitor of autophagy. Autophagy expression in cells treated with chemotherapeutic agent was measured as mentioned above. The level of apoptosis of SP cells with or without 3-MA was determined by flow cytometric analysis. The cells were then incubated in 5 ml binding buffer containing 5 ml Annexin V and 5 ml propidium iodide. The cells were gently vortexed and assayed with an Annexin V-FITC apoptosis detection kit according to the manufacturer’s instructions.
Statistical analysis
Data were presented as the mean ± SD. Statistical significance (P<0.05) was evaluated by the Student’s t-test.
Results
Identification of SP cells in lung cancer
SP cells were separated from A549 lung cancer cells by their fluorescence profiles in a dual wavelength analysis by FACS. The characteristic tails isolated from the complete population were identified.
The percentage of SP cells analyzed by FACS was 1.10% (Fig. 1A). After preincubation with verapamil which inhibited ABC transporters for 90 min, the percentage of SP cells decreased to 0.20% (Fig. 1B). This result showed that Hoechst 33342 exclusion was verapamil-sensitive. The images of SP cells were captured by confocal microscopy (Fig. 2).
Sphere formation
The floating spheres of SP cells were observed after 3–4 days of seeding, and the primary spheres containing >30 cells were observed after 10–14 days (Fig. 3A and B). The amount and formation speed of the secondary sphere were similar to those of the primary sphere (Fig. 3C), suggesting that SP cells have self-renewal ability. However, non-SP cells could not be propagated under the same conditions.
Cell proliferation and colony formation
We performed CCK-8 analysis to compare the cell proliferation of SP with non-SP cells. A significant difference in cell proliferation between SP and non-SP cells was observed after 5 days of culture (P<0.05) (Fig. 4A). In addition, in the colony formation assay, there was a statistically significant difference between SP and non-SP cells over 10–14 days (P<0.05) (Fig. 4B).
Expression of stem cell genes
MDR1 and ABCG2, which belong to ABC transporters, contribute to the chemoresistance of SP cells. Compared to non-SP cells, we found that the expression of MDR1 and ABCG2 in SP cells was significantly upregulated. OCT-4, an embryonic stem cell biomarker, was also highly expressed in SP compared with non-SP cells. The results indicated that SP cells from A549 lung cancer cells had some features of cancer stem cells (Fig. 5).
Tumor formation
To determine the tumorigenic potential of lung cancer SP cells in vivo, SP and non-SP cells were subcutaneously injected into 6- to 7-week-old nude mice. Each mouse was injected with different density of SP and non-SP cells individually. After 8 weeks, almost all of the mice injected with SP cells at densities of 104–103 cells formed tumors, whereas a smaller number of mice injected with non-SP cells at a density of 104 cells formed tumors. Additionally, the tumor diameters were apparently smaller than those in SP cells with an equal cell number (Fig. 6).
Autophagy expression in SP and non-SP cells
MDC staining was performed to detect the autophagolysosomes. We observed that the number of autophagolysosomes in SP cells was less than that in non-SP cells by confocal laser microscopy (Fig. 7). The expression of Beclin-1 and LC3-II, both of which were involved in the autophagic process, was lower in SP cells than that in non-SP cells (Fig. 8).
Autophagy provides resistance against chemotherapy in SP cells
A significant increase in autophagic response towards the chemotherapeutic drug, cisplatin, was observed in SP cells. Autophagosomes were found to be markedly increased in the presence of cisplatin. Compared to SP cells without treatment of cisplatin, the expression of Beclin-1 and LC3-II in SP cells treated with cisplatin was markedly upregulated. In addition, the rate of apoptosis in SP cells with cisplatin was (4.2±0.7)%, while it increased up to (31.4±2.3)% in the presence of 3-MA, which is an inhibitor of autophagy (Fig. 9).
Discussion
The CSCs hypothesis suggests that cancer may be maintained by CSCs that possess the properties of self-renewal, multipotency and carcinogenesis (14). Recent evidence has shown that CSCs are the root of recurrence, metastasis and resistance to the chemotherapy of cancer (15,16). Therefore, it is suggested that targeting CSCs in cancer provides a novel effective therapeutic strategy to eradicate them completely. Regarding the methods of isolating CSCs, identification of specific markers for CSCs is the ideal method. It is reported that CSCs have been identified by specific markers in several solid tumors, including breast (17), prostate (18) and colon cancer (19), and melanoma (20). However, specific markers of CSCs in malignant tumors remain to be identified. Furthermore, the so-called ‘CSCs’, which had differentiated surface markers, were not considered real CSCs, as they had already differentiated (21,22).
Due to the absence of specific markers of CSCs in most malignant tumors, sorting SP cells using FCM by a dye Hoechst 33342 has become the main method of investigating CSCs. SP cells efflux the dye Hoechst 33342 through an ABC membrane transporter which is responsible for chemoresistance and is also thought to possess stem cell characteristics in several types of cancer (23). In the present study, we isolated SP cells from the A549 lung cancer cell line by flow cytometry and then verified the cancer stem cell-like properties of SP cells in vitro and in vivo.
It is reported that CSCs have an anchorage-independent growth ability and sphere formation assays can be used to check the self-renewal ability of cells in vitro (24). In the present study, the formation of the primary and secondary floating sphere showed that SP cells isolated from A549 lung cancer cells possessed self-renewal ability. Infinite proliferation and tumorigenic abilities are notable features of CSCs (25). Compared to non-SP cells, the fact that single cells became anchorage-independent more rapidly and formed colonies showed that SP cells had malignant and tumorigenic abilities. These findings suggest that SP cells from lung cancer cells possessed CSCs-like properties.
The expression of MDR1 and ABCG2, which belong to the ABC transporter superfamily, were upregulated in SP cells when compared with that in non-SP cells. This result was consistent with the higher chemotherapeutic resistance of SP cells. OCT-4 is a transcriptional activator belonging to the POU family, and it is also an important regulator of self-renewal and differentiation in stem cells. The overexpression of these representative stem cell genes in SP cells suggested that the SP cells characterized the properties of CSCs.
For the in vivo experiments, SP and non-SP cells were injected into nude mice subcutaneously. SP cells formed tumors after 8 weeks at densities of 103 and 104, whereas non-SP cells only formed tumors in some mice at a density of 104, and the tumors were much smaller than that of the SP group. Of note was that some non-SP cells also formed tumors in some nude mice, suggesting that although CSCs were enriched in SP cells, non-SP cells also contain some CSCs.
Autophagy is a pivotal process for cell survival by decomposing unessential proteins or impaired cell organelles and utilizing them as energy when cells suffer from malnutrition, exposure to toxic materials, hypoxia and virus infection (26). If the stresses continue, this process leads to autophagic cell death (27). Notably, autophagy has two contradictory aspects in that it acts as a mechanism of cell survival and death (28). In a previous experiment, using A549 lung cancer cells, we identified that inhibition of autophagy was capable of promoting the apoptosis induced by chemotherapeutics. In the present study, we found that the expression of autophagosomes was much lower in SP cells than that in non-SP cells. In addition, Beclin-1 and LC3-II, both of which were involved in the autophagic process, also showed a lower expression in SP cells compared to that in non-SP cells. However, the autophagy level was found to be markedly increased in the presence of cisplatin and inhibition of autophagy enhanced the effects of apoptosis induced by the chemotherapeutic agent, cisplatin, in lung cancer SP cells. Thus, autophagy is a potential therapeutic target for lung cancer.
In conclusion, SP cells from the A549 lung cancer cell line can be effectively sorted using FACS with Hoechst 33342. SP cells possess the properties of CSC, such as increased proliferation, higher clonogenicity, and stronger tumorigenicity. Moreover, SP cells are more resistant to chemotherapy and inhibition of autophagy enhances the effects of apoptosis induced by cisplatin. Therefore, these findings collectively suggest that SP cells from the A549 lung cancer cell line have cancer stem cell-like properties and autophagy may be an attractive therapeutic target for human lung cancer.