Background
Lung cancer is one of the most common malignancies worldwide and it ranks as the most frequent cause of cancer-related deaths especially in urban areas of China [
1,
2]. The most common subtypes of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). And the latter is the most aggressive form of lung cancer, with a high invasive capacity and greater tendency for development of early metastasis [
3]. Despite all kinds of conventional therapeutic regime applied in clinical treatment, lung cancer has a dismal 5-year survival rate due to failure of currently available therapeutic approaches and finally resulting in disease relapse [
4].
Researchers frequently attributes the therapy resistance of lung cancer to cancer stem cells(CSCs) within the tumors. Increasing evidence has indicated that many types of solid cancer, including lung cancer, are originated from and maintained by a minor subpopulation of CSCs [
5,
6]. These cells possess the capacity of continuous self-renewal, differentiation and initiating tumor formation as well as show increased resistance to radiation and chemotherapy. CSCs are considered as the origin of tumor relapse.
Recently, various methods based on specific cell surface markers, the capacity of cells to exclude membrane permeable dyes or the ability of tumor sphere formation for single cell in suspension in serum-free medium have been used for CSCs isolation and characterization [
7‐
9]. Similar to other solid tumors, several putative surface markers for lung CSC(LCSCs) have been identified, including CD133 and CD44 [
6,
10‐
12]. Eramo et al. found the existence of a rare population of undifferentiated cells expressing CD133 in small cell and non-small cell lung cancer for the first time. These minor population of CD133+ tumorigenic cells able to self-renew and give rise to an unlimited progeny of differentiated tumor cells are named lung cancer stem cells (LCSCs) [
10]. However, another study by Meng et al. found out that both the CD133+ and CD133- subpopulations from SCLC cells contain similar numbers of cancer stem cells, which suggest that CD133 alone cannot be used as a stem cell marker for the SCLC [
7]. In addition, Jiang et al. also demonstrated that Aldehyde dehydrogenase 1 (ALDH1) is a tumor stem cell-associated marker in lung cancer [
8]. Taken together, although no certain specific cell markers for LCSCs have been defined yet, we think that the suspended culture with stem cell-conditioned medium can be at least used as a crucial approach for enrichment of LCSCs. Therefore, developing new chemotherapy agents that can target these LCSCs has profound implications for cancer chemoprevention and therapy.
Fructus Viticis (Manjingzi is the Chinese name), the dried ripe fruit of Vitex trifolia L. (family Verbenaceae), is a traditional Chinese medicine possessing a broad range of pharmacological activities [
13‐
15]. Fructus Viticis contains large amounts of flavonoids, including casticin, luteolin, apigenin, isoorientin, hesperidin, isovitexin and so on [
16‐
18]. Accumulating studies have substantiated the anti-tumor properties of these flavonoids in a diverse range of human cancer cells and their xenograft models in mice [
19,
20]. FVTF(Fructus Viticis Total Flavonoids), an independent innovation of our laboratory, is a candidate Chinese medicine preparation extracted from Fructus Viticis (National invention application No. ZL 201210591146.9).
In the present study, we obtained the CD133+ sphere-forming cells (SFCs) from NCI-H446 cells by employing CD133+ magnetic separation and stem cell-conditioned medium culture method. Furthermore, we confirmed the LCSLCs characteristic of SFCs by compared the self-renewal capacity, stem cell markers expression levels, and tumorigenicity of SFCs and non-sorting cells, namely parental cells. The aims of this study were to investigate whether FVTF could inhibit the characteristics of LCSLCs and the potential mechanisms involved.
Methods
Drugs and reagents
The Fructus Vititcis was purchased from Yuling Zhonggui Pharmaceutical Company (Yuling, Guangxi province, China). Prof. Bing-Qing Zhao (Department of Pharmacy, Medical College of Hunan Normal University) identified the specimens. A voucher specimen has been deposited in the Department of Pharmacy, Medical College of Hunan Normal University (No. MJZ-001). Fructus Viticis Total Flavonoids(FVTF) was extracted and prepared from Fructus Viticis according to the patent specification (National invention application of China No. ZL 201210591146.9), present as brown powder. The measured amounts of total flavonoids in drying FVTF were 53 % calculated based on rutin.
DMEM medium(High glucose), DMEM/F12 medium, fetal bovine serum, 0.25 % trypsin(containing EDTA) and phosphate buffered saline (PBS) were purchased from Hyclone(Logan, UT, USA). Recombination human fibroblast growth factor (bFGF), epidermal growth factor (EGF) and B27 were purchased from eBioscience. Hematoxylin and Eosin Staining Kit, Matrigel (BD Biosciences, USA). Cell lysis buffer, trypan Blue, penicillin and streptomycin (Invitrogen Life Technologies) and bovine serum albumin were purchased from Hunan Clonetimes Biotech Co., Ltd. (Changsha, Hunan). Human insulin were purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd. Mouse monoclonal antibodies anti-CD44, ALDH1, Bmi1, Nanog, Oct4, Twist, Snail, p-Akt (Ser473), Akt, β-actin were from Cell Signaling Technology (Beverly, MA, USA).
Cell culture
Human small cell lung cancer (SCLC) cell line NCI-H446 was purchased from Keygen biotechnology Co., Ltd. (NanJing, China), and was maintained in DMEM (High glucose) supplemented with 10 % fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin in an incubator containing 5 % CO2 at 37 °C.
Magnetic separation of CD133+ cells
The CD133+ positive cells were sorted from NCI-H446 cell line using magnetic separation (Miltenyi Biotec, Germany) according to the manufacturer’s instruction. Briefly, a single-cell suspension was firstly prepared and the cell number were determined before magnetic labeling. Cells were collected by centrifuge at at 300 × g for 10 min and the cell pellet were resuspended in 60 μL of buffer per 107 total cells. 20 μL of FcR Blocking Reagent and CD133 MicroBeads were added per 107 total cells, respectively. After mixing well and incubation at 2-8 °C for 15 min, cells were washed and resuspended up to107 cells in 500 μL of buffer. The cell suspension was finally applied onto a MACS Column, and the unlabeled cells (CD133−) and magnetically labeled cells (CD133+) were collected repectively. Thereafter, the sorted CD133+ cells were seeded in ultra-low attachment 6-well plates (Corning Inc., NY, USA) for amplification culture with stem cell-conditioned medium.
Flow cytometry analysis
The sorted CD133+ cells, CD133− cells and unsorted parental NCI-H446 cells were washed with PBS, and were adjusted to the single cell suspension at a density of 1 × 105 cells/mL, respectively. Cells were incubated with PE-conjugated anti-human CD133 antibody (Biolegend, San Diego, CA, United States) for 30 min at 4 °C in the dark, and mouse IgG2b-PE was used as an isotype control. Finally, The cell suspension was applied onto a flow cytometry analysis of the percentage of CD133+ cells.
The self-renewal capacity of cells was evaluated using suspension culture in ultra-low attachment 6-well plates (Corning Inc., NY, USA) with stem cell-conditioned medium [
20]. Single cell suspensions were prepared with stem cell-conditioned medium and were loaded into ultra-low attachment 6-well plates (2 × 10
3cells/ml). After culture for 6 days, the number of tumor spheres formed were counted and pictured under a phase-contrast microscope (Nikon Eclipse TE2000-S). The sphere forming rate was calculated as the total number of tumor spheres formed/the total number of cells inoculated × 100 %. For drug-intervention experiment, different concentrations of FTVF were added to the medium prior to cells were inoculated. For secondary sphere formation assay, tumor spheres derived from CD133
+ NCI-H446 cells were digested and prepared into single cell suspensions. After that, cells were seeded into ultra-low attachment 6-well plates at a density of 1 × 10
3cells/mL, and no drugs was added in the procedure of secondary sphere formation assay.
MTT assay
Parental NCI-H446 cells and CD133
+ SFCs were seeded at a density of 5 × 10
3 cells/well in a 96-well plate pre-coated with 0.6 % agarose, respectively. The procedure for MTT assay and the calculation of relative cell proliferation inhibition rate have been previously described [
19]. The IC50 value was calculated using the Prism software (GraphPad Software, San Diego, CA).
In vitro cell migration and invasion assays
The migration and invasion assays were performed using 24-well uncoated or Matrigel (BD Biosciences, United States) coated Transwell chambers with 8.0-μm pore polycarbonate membranes (Corning Coster, Cambridge, MA, USA) as previously described [
21]. Briefly, 600 μL DMEM with 10 % FBS was added to the 24-well culture plate (lower chambers). Then the Transwell chambers were inserted into the 24-well plate, and cells were resuspended in a serum and growth factor-free DMEM and loaded into chambers (1 × 10
4 cells/100 μl). Following incubation at 37 °C, cells that migrated or invaded to the lower surface of the membrane were fixed and stained with hematoxylin-eosin or Wright-Giemsa. Finally, the stained cells were digitally imaged and counted under an optical microscope (OLYMPUS IX51, Japan). For drug-intervention experiment, cells were pretreated with different concentrations of FTVF for 24 h prior to the transwell chamber assay.
Western blot analysis
Western blot procedures were performed as previously described [
22]. The following primary antibodies were used: Mouse monoclonal antibodies anti-CD44, ALDH1, Bmi1, Nanog, Oct4, Twist, Snail, p-Akt (Ser473) and Akt. β-actin was used as an internal control. Signals were detected using Enhanced chemiluminescence Kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA).
Nude mice tumorigenicity assay
The SPF male Balb/c-nu mice were obtained from the Hunan Slac Jingda Laboratory Animal Co., Ltd. (Yueyang, Hunan). All animal studies in this study were performed according to the experimental animal breeding and use guide of Hunan Laboratory Animal Management Committee after approved by the Ethical Committee of Hunan Normal University. 18 SPF male Balb/c-nu mice were randomly devided into three groups (6/group). CD133+ cells and parental cells in PBS were mixed 1:1 with Matrigel (BD Biosciences, San Jose, CA) and were injected subcutaneously into the flanks of the animals, respectively. The numbers of cells inoculated in three groups were 1 × 102, 1 × 103 and 1 × 104/mouse for CD133+ SFCs, and 1 × 104, 1 × 105 and 1 × 106/mouse for parental cells, respectively. The animals were maintained in accordance with standard guidelines for about 2 months for tumor appearance. The size of tumor nodules were measured using an caliper, and the volume was calculated using V (mm3) = L (the longest diameter) × W (the shortest diameter)2 × 0.5. At the end of experiment, the tumors were excised, weighed and divided into pieces to be fixed in neutral buffered formalin and embedded in paraffin and sectioned for a routine histological evaluation using hematoxylin and eosin (H&E) staining or for immunohistochemical analysis of CD44 and CD133.
Immunohistochemical examination
The xenografted tumor tissuses were fixed in 10 % formalin, embedded in paraffin and sectioned for immunohistochemical analysis. For immunohistochemical staining, the deparaffinized sections were incubated with 3.3 % H2O2 for 10 min to block endogenous peroxidase activity. The sections were then blocked with Tris Buffered Saline (TBS) containing 5 % normal goat serum for 30 min. After incubated with diluted corresponding primary antibodies (1:200 for mouse anti-CD133 antibody and 1:250 for mouse anti-CD44 antibody) and a secondary antibody, the signals were detected using an EnVision system (Dako, Carpinteria, CA, USA) and counterstained with hematoxylin .
Statistics analysis
Results are shown as mean ± SD. Analysis of variance was performed with SPSS 15.0 software for Windows using one-way ANOVA and pair-wise comparison with t test. P < 0.05 was considered statistically significant.
Discussion
Despite all kinds of efforts exploited to combat lung cancer, this disease remains the most frequent cause of cancer-related deaths in China. It is now generally accepted that LCSLCs were the determining tumorigenic cells responsible for lung cancer initiation, development, metastasis and relapse after chemotherapy. Therefore, targeted eradication of LCSLCs is extremely vital for improving the prognosis or even recovery of lung cancer.
In the present study, we isolated, enriched and obtained the CD133+ SFCs from human lung cancer NCI-H446 cells, by CD133+ magnetic separation and stem cell-conditioned medium suspension culture method. The following results confirmed that this subpopulation of cells exhibited higher in vitro self renewal, invasion and tumorigenicity capacity over parental cells, accompanied by an upregulated expression level of stem cell markers, self-renewal and invasion related transcription factors. Collectively, these SFCs exerted stem-like cell characteristics and were defined as LCSLCs.
Increasing new emerging data have reported that transcription factors Bmi1, Nanog and Oct4 have been implicated in regulating self-renewal and multi-directional differentiation potential of CSCs and adult stem cells. Park IK et al. has reported that Bmi1, a member of the polycomb group proteins, is essential for maintenance of adult self-renewing haematopoietic stem cells [
23]. Another study by Abdouh M et al. also revealed that PcG proteins are involved in Glioblastoma multiforme (GBM) tumor growth and required to sustain cancer initiating stem cell renewal [
24]. Nanog and Oct4 are the core transcription factors that maintaining the self-renewal and pluripotency of embryonic stem cells [
25‐
28]. It has been reported that coexpression of Oct4 and Nanog promotes metastasis of lung adenocarcinoma by inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiation [
29]. In this study, we found that Bmi1, Nanog and Oct4 were highly expressed in SFCs derived from NCI-H446 cells compared to that in parental cells, as confirmed by western blot analysis. It indicated that the SFCs possessed self renewal and pluripotency properties.
It is generally accepted that most cancer related-deaths are attributed to the local invasion and distal migration, but not the primary tumor itself [
30,
31]. CSCs were considered to play a pivotal role in the early metastasis of many kinds of human malignant tumors including lung cancer. In our study, it was demonstrated by Transwell assays that SFCs derived from NCI-H446 cells exhibted higher invasion capacity in vitro than parental cells. These results revealed that SFCs possessed high invasion and metastatic potential. The invasion related protein Snail was one member of the zinc-finger transcription factor Snail superfamily. It has been reported that Snail can directly or indirectly mediated epithelial-mesenchymal transition (EMT) which results in tumor cell invasion and metastases. In clinical practice, overexpression of Snail is associated with poor prognosis and increased invasioness in several type of cancer patients [
32‐
34]. Twist1, which belonged to the basic Helix-Loop-Helix (bHLH) transcription factor family, is a highly evolutionally conserved transcription factor firstly found in
Drosophila and initially involved in embryo development by contributing to cell migration. A study from Mikheeva et al. has reported that TWIST1 promotes invasion through activation of mesenchymal molecular and cellular changes in human glioblastoma [
35]. Li et al. has revealed that Twist1 depletion completely blocked the mesenchymal transformation induced by Adriamycin [
36]. Our previous study also found that Twist signaling involved in maintaining self-renewal and invasion properties of liver cancer stem cells. In this study, we showed that the EMT-related transcription factors Snail and Twist1 were highly expressed in SFCs as compared to that in parental cells. Although no gene expression regulation measures were further taken, we have reasons to speculate that the high invasion capacity of SFCs may be related to its high expression level of Snail1 and Twist.
Since increasing studies have proved that there exists a plenty of active compounds with anti-cancer properties in some phytomedicines, extracting and isolating anti-cancer agents from naturally-occurring plants becomes one of the main approaches for new anti-tumor drugs discovery. In the present study, we demonstrated that the candidate Chinese medicine preparation FVTF could relative selectively inhibit the proliferation and self-renewal, and suppress migration and invasion of LCSLCs in vitro. In agreement with above findings, we also found that FVTF down-regulated the protein expression of stem cell markers (CD133, CD44 and ALDH1), self-renewal associated transcription factors (Bmi1, Nanog and OCT4) and invasion associated transcription factors (Twist1 and Snail1) in a dose-dependent manner. Furthermore, we demonstrated that the inhibitory effects of FVTF on LCSLCs characteristics appeared to be mediated through down-regulating expression of p-Akt.
Akt, also known as protein kinase B(PKB), is a multifunctional serine-theonine protein kinase involved in a variety of biological functions in cells. A recent study has reported that Akt activation in Hair follicle stem cells (HF-SCs) results in increased tumor development and malignant transformation [
37]. Chen et al. showed that Akt could phosphorylate Oct4 at threonine 228 to accelerate its degradation. And Akt mediated phosphorylation of special AT-rich sequences binding protein 1 (SATB1) played a key role in inhibiting Nanog expression [
38]. Another study by Wang et al. found that AKT/β-catenin/Snail signaling pathway is mechanistically involved in acquisition of cancer stem cell-like properties and EMT features in cisplatin-resistant A549 cells [
39]. In this study, we also showed that the constitutively activation of Akt was involved in maintaining self-renewal of LCSLCs. And the use of FVTF or LY294002 could significantly decrease the phosphorylation level of Akt in LCSLCs. Interestingly, we also found that LY294002 could act alone or synergize with FVTF to suppress the characteristics of LCSLCs. Therefore, it is reasonable to speculate that FVTF may inhibited the phosphorylation of Akt by a pathway different from the way that LY294002 acted in.
Taken together, our results have demonstrated that FVTF can inhibit characteristics of LCSLCs derived from NCI-H446 cell line. And the mechanisms is probably associated with down-regulating expression of p-Akt in LCSLCs, which lead to downregulation of the expressions of stem cell markers, self-renewal associated transcription factors and invasion associated transcription factors. However, based on the limitation of conducting these experiments in a single cell line derived LCSLCs, future efforts would be made toward confirming the therapeutic effects of FVTF in more cell lines as well as in animal model.
Acknowledgments
Not applicable.