Acta Biochimica et Biophysica Sinica Advance Access published November 17, 2013 Acta Biochim Biophys Sin 2013, xx: 1 – 7 | ª The Author 2013. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt123.
Original Article
Casticin suppresses self-renewal and invasion of lung cancer stem-like cells from A549 cells through down-regulation of pAkt Fei Liu1†, Xiaozheng Cao1†, Zhihong Liu2, Hui Guo2, Kaiqun Ren1, Meifang Quan1, Yuan Zhou1, Honglin Xiang1, and Jianguo Cao1 * 1
College of Medicine, Hunan Normal University, Changsha 410013, China Department of pathology, Hunan Provincial Tumor Hospital, Changsha 410013, China † These authors contributed equally to this work. *Correspondence address. Tel/Fax: þ86-731-88912434; E-mail: [email protected] 2
Keywords lung cancer; casticin; cancer stem cells; pAkt; therapeutic action Received: May 22, 2013
Accepted: September 2, 2013
Introduction Lung cancer is the most common cause of cancer deaths worldwide for its high incidence and mortality. Globally, more than one million new lung cancer cases were reported each year [1,2]. Lung cancer is generally subdivided into
small cell lung cancer and non-small cell lung cancer (NSCLC). The 5-year survival for NSCLC is less than 9% in developing countries [2], and no more than 15% in USA [3]. So it is necessary to explore the novel therapeutic approaches for improving life quality of patients with lung cancer including NSCLC. Recently, more and more researchers are focusing on cancer stem cells (CSCs), trying to find new breakthrough point for curing cancer. The CSCs possess self-renewal capacity and differentiation potential, and can reconstruct the phenotypic and histologic heterogeneity of its parent tumor when transplanted in vivo [4]. The CSC theory presumes that the tumor carcinogenesis, recurrence, and resistance are caused by the existence of a small subpopulation of CSCs. Recent studies are mainly involved in characterizing CSCs from different tumor cell lines or solid tumor tissues including glioblastoma, melanoma, breast cancer, lung cancer, etc., and their crucial signal molecules [5–8]. CSCs can be enriched or identified using cell surface markers via flow cytometry and immunomagnetic beads sorting system [9,10], or sphere-forming assay [4,11] in selective serum-free medium. In NSCLC, CSCs are commonly characterized as a subpopulation of cells expressing CD133 [10], or aldehyde dehydrogenase (ALDH) [12,13] or as a side population [14] of cells capable of effluxing Hoechst dye. Identification of CSCs in tumor cells is important, but studies on this area are just the beginning. What is more important is to find the effective therapeutic strategies, including screening of novel drugs targeting CSCs. Casticin (5,30 -dihydroxy-3,6,7,40 -tetramethoxyflavone) is a main active ingredient of Fructus Viticis Simplicifoliae, which is often used as the traditional Chinese medicine for the treatment of certain types of cancer. Studies have shown that casticin possesses extensive anticancer bioactivity for cancers including lung cancer [15], cervical cancer [16], leukemia [17,18], colon cancer [19], hepatocellular carcinoma Acta Biochim Biophys Sin (2013) | Page 1
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A subpopulation of cancer stem cells is recognized as the cause of tumorigenesis and spreading. To investigate the effects of casticin (5,30 -dihydroxy-3,6,7,40 -tetramethoxyflavone), derived from Fructus Viticis Simplicifoliae, on lung cancer stem cells, we isolated and identified a subpopulation of lung cancer stem-like cells (LCSLCs) from non-small-cell lung carcinoma A549 cells with the features including selfrenewal capacity and high invasiveness in vitro, elevated tumorigenic activity in vivo, and high expression of stemness markers CD133, CD44, and aldehyde dehydrogenase 1 (ALDH1), using serum-free suspension sphere-forming culture method. We then found that casticin could suppress the proliferation of LCSLCs in a concentration-dependent manner with an IC50 value of 0.4 mmol/L, being much stronger than that in parental A549 cells. In addition, casticin could suppress the self-renewal and invasion of LCSLCs concomitant with decreased CD133, CD44, and ALDH1 protein expression and reduced MMP-9 activity. Further experiments showed that casticin suppressed selfrenewal and invasion at least partly through down-regulation of Akt phosphorylation. In conclusion, casticin suppressed the characteristics of LCSLCs, suggesting that casticin may be a candidate compound for curing lung cancer via eliminating cancer stem cells.
Casticin suppresses lung cancer stem-like cells
cells [20], and so on [21,22]. We have found that casticin could inhibit self-renewal of glioma stem-like cells [23]. However, the effects of casticin on lung cancer stem-like cells (LCSLCs) and its mechanisms remain elusive. In this study, we enriched and identified LCSLCs from NSCLC A549 cells, and found that casticin could suppress the self-renewal and invasion of LCSLCs, accompanied with a reduced CD133, CD44, and ALDH expression. The underlying mechanisms were associated with the decrease of phosphorylation Akt, which was reported to play a crucial role in the maintenance of CSC features.
Materials and Methods
Matrigel invasion assay Two thousands cells incubated in serum-free medium were added on the top of the transwell chamber coated with fresh Matrigel (Gibco). The medium supplemented with 10% FBS, as the chemical inducing agent, was added to the bottom of the transwell chamber. After incubation for 48 h, the cells on top of the chamber were scraped off using a cotton swab. And the cells in the bottom of the chamber were fixed with methanol and stained with Wright staining solution. The cells invading through the membrane were counted under an optical microscope.
Sphere-forming and self-renewal assay To obtain sphere cultures, cells were plated at a density of 5 103 cells/well in 6-well ultra-low plates (Corning, Acton, USA) containing serum-free medium DMEM/F12 (Gibco, Carlsbad, USA), supplemented with commercial hormone mix B27 (Gibco), 20 ng/ml EGF (PeproTech, Rocky Hill, USA), 10 ng/ml bFGF (PeproTech), 0.4% bovine serum albumin (Gibco), 4 mg/ml insulin (Gibco), 100 U/ml penicillin and 100 U/ml streptomycin at 378C. After being cultured for 6 days, the lung cancer spheres were collected, dissociated into single cell suspension and resuspended in fresh medium for serial subcultivation every 6 days. To investigate the self-renewal capacity of lung cancer sphere-forming cells (SFCs), single cell suspension prepared from the lung cancer sphere was diluted to 500 cells/ml. Two microliters of the single cell suspension was plated in 96-well ultra-low plates containing 150 ml serum-free medium per well. Wells containing no cells or more than one cell were excluded, and those with one cell were marked and monitored daily under a microscope (Nikon Eclipse TE2000-S, Nikon, Japan) for 6 days and the colonies were counted. To investigate the effects of casticin on self-renewal of SFCs, single cell suspension of SFCs was plated at a density of 2 103 cells/ml in 6-well ultra-low plates. Different concentrations of casticin (1.0, 5.0, and 10.0 mM) were added to medium. After being cultured for 6 days, the colonies were counted under a microscope.
MMP-9 activity assay The SFCs were plated at a density of 1 106 cells/well in 6-well ultra-low plates containing serum-free medium. After 24 h incubation, the medium was replaced with serum-free medium containing different concentrations of casticin (1.0, 5.0, 10.0 mM), cultured for additional 72 h. Then the medium was collected and subject to MMP-9 activity assay using human active MMP-9 fluorokine E kit (R&D Systems, Minneapolis, USA) according to manufacturer’s instructions. The activity unit was defined as ng/ml/106 cells.
MTT [3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay The SFCs or parental cells were plated at 5000 cells/well in 96-well ultra-low plates. The cells were first incubated with
Western blot analysis The cells were lysed using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2 mM EDTA, 0.2% NP-40, 10% glycerol, 1 M b-Me, 1 mg/ml aprotinin, 0.5 mg/ml aprotinin
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Tumorigenicity assay The SFCs or parental cells were injected subcutaneously into the back of 4-week-old BALB/C-nude mice (supplied by the Shanghai Experimental Animal Center, Chinese Academy of Sciences, Shanghai, China). The mice were reared for 2 months, and the tumor growth and tumorigenic time were examined visually. Tumor volumes were calculated in accordance with the formula: V (transplanted tumor volume, mm3) ¼ L (longest diameter, mm) W (minimum diameter, mm)2 0.5. At the end of experiment, the mice were sacrificed under deep anesthesia with pentobarbital. The tumors were then dissected, weighed and fixed with 10% neutral formalin. Paraffin sections were prepared following routine procedures for H&E staining.
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Cell culture Human lung cancer A549 cell line was purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China), cultured in RPMI-1640 (HyClone, Logan, USA), supplemented with 10% fetal bovine serum (FBS) at 378C in 5% CO2.
casticin at the indicated concentrations for 48 h, and then incubated with 5 mg/ml MTT for 4 h. Lastly, 100 ml of dimethyl sulfoxide (DMSO) was added to each well, and the absorbance at 570 nm (A570) each well was detected on a plate reader (Bio-Tek EXL-800, Winooski, USA). The relative cell proliferation inhibition rate ¼ (1— experimental group A570 mean/control group A570 mean) 100%. IC50 (50% inhibiting concentration) was calculated by Prism (GraphPad Software, San Diego, USA).
Casticin suppresses lung cancer stem-like cells
Statistical analysis Data were presented as the mean + standard deviation. To assess the statistical significance of differences, student’s t test was performed. P , 0.05 was regarded as statistically significant.
Results Lung cancer SFCs derived from A549 cell lines have the self-renewal capability In order to enrich LCSLCs from human lung cancer A549 cell line, stem cell conditioned medium suspension culture method was used. The results showed that A549 cells could form non-adherent sphere which is called lung cancer SFCs (Fig. 1A2). The results suggested that LCSLCs exist in human lung cancer A549 cells. We next performed the SFCs subculture and tumor sphere formation test. It was found that the single cell of SFC from lung cancer A549 was able to form a new sphere (Fig. 1B), suggesting that the SFCs from A549 cells have the ability of self-renewal. Lung cancer SFCs overexpress stem cell markers Furthermore, we investigated the expressions of CSC biomarker CD44, CD133, and aldehyde dehydrogenase 1 (ALDH1) in SFCs using western blotting. The results demonstrated that, compared with human lung cancer parental cells, the CD44, CD133, and ALDH1 protein expression levels were apparently increased in their SFCs (Fig. 1C). Lung cancer SFCs possess higher tumorigenicity In addition, the xenograft tumor model in nude mouse was used to investigate the tumorigenicity of A549 cells and
Figure 1. Identification of LCSLCs from lung cancer A549 cells Parental cell A549 (A1) and its SFC enriched by stem cell conditioned medium suspension culture method (A2). The single cell of SFC formed a new sphere (B). The CD44, CD133 and ALDH1 protein expression levels in SFC and their PCs (C). The tumorigenicity (D) and immunohistochemical characteristics (E) of A549 cells and its SFC xenograft tumor in Balb/c-nu immunodeficient mice. PC: Parental cell.
their SFCs in Balb/c-nu immunodeficient mice. The results showed that 1 104 A549 SFCs were enough to form tumors in vivo (Fig. 1D), and the tumors have similar immunohistochemical characteristics with its parental cell xenograft tumor (Fig. 1E). However, in the same model, at least 1 106 A549 parental cells were required to induce stable tumor formation (Table 1). Our data indicate that the SFCs from A549 cell line have higher tumorigenicity in vivo than parental cells. The above data confirmed that SFCs from human lung cancer A549 cell line cells possessed characteristics of CSC. Therefore, we can, at least, deduce that the lung cancer SFCs prepared by us belong to LCSLCs, and the LCSCs were possibly enriched in them.
Casticin inhibits the self-renewal capability of LCSLCs The unlimited proliferation potential is an important feature of CSCs [24]. Genistein is a natural polyphenolic compound that can preferentially inhibit the proliferative activity of the pancreatic CSCs than parent cells [23]. Our results Acta Biochim Biophys Sin (2013) | Page 3
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aldehyde peptide, 0.1 mM Na3VO4, 0.5 mM 4 NPP, 0.5 mM NaF, and protease inhibitors), and the total cell extracts were prepared. The protein content was measured using the Bradford method. The proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. After being blocked with 5% non-fat milk in phosphate buffered saline containing Tween-20 (PBST), the PVDF membrane was incubated using the following mouse anti-human primary monoclonal antibodies: anti-pAkt (Ser473) (1 : 500; Cell signaling, Beverly, USA), anti-Akt (1 : 2000; cell signaling), anti-CD44 (1 : 500; cell signaling), anti-CD133 (1 : 500; cell signaling), antiALDH1 (1 : 500; cell signaling), and anti-b-actin (1 : 200; Sigma, St. Louis, USA), with slight shaking at 48C overnight. Then the membrane was incubated with peroxidase-labeled secondary antibody for 1 h, and the signal was detected using an enhanced chemiluminescence kit (Amersham Biosciences, Buckinghamshire, UK) according to manufactures’ protocols.
Casticin suppresses lung cancer stem-like cells
showed that casticin (0.1, 1, 3, 10, 30 mM) could inhibit the proliferation of human lung cancer SFCs in a dosedependent manner (Fig. 2A), and the IC50 of casticin on A549 cells and their SFCs were 14.3 and 0.4 mmol/L, respectively, suggesting that casticin is able to preferentially suppress the proliferation activity of LCSLCs. Furthermore, casticin (1, 5, 10 mM) could reduce the size and number of SFCs from A549 cells in a dose-dependent manner (Fig. 2B,C), suggesting that casticin can suppress the self-renewal of LCSLCs.
Table 1. Xenotransplantation of human lung cancer A549 cells and its SFCs into Balb/c-nu immunodeficient mice
Cell
Inoculum size Tumor incidence Latency period (d)
A549 cells 1 104 1 105 1 106 SFCs 2 103 1 104 1 105
0/4 0/4 2/4 2/4 4/4 4/4
– – 31 29 19 11
Casticin reduces CD133, CD44, and ALDH1 expression in LCSLCs Western blot analysis showed that casticin (1, 5, 10 mM) reduced CD133, CD44, and ALDH1 protein expression in SFC from A549 cells, in a dose-dependent manner (Fig. 3A). It suggests that casticin can decrease CSC marker expression in LCSLCs. Casticin inhibits the self-renewal of LCSLCs via down-regulation of pAkt Western blot analysis showed that the phosphorylation levels of Akt in SFCs from A549 cells were higher than those in A549 cells (Fig. 3B). Casticin could decrease phosphorylated Akt ( p-Akt) expression in the SFCs in a dosedependent manner (Fig. 3C). Akt inhibitor LY294002 (5, 10, 20 mM) could effectively reduce phosphorylation of
Figure 2. Effects of casticin on LCSLCs Casticin inhibited proliferation (A) and reduced the size (B) and number (C) of SFC from A549 cells. Casticin suppressed the invasion of SFC from A549 cells (D) and (E). Casticin decreased MMP-9 activity (F). *P , 0.05 vs. 0.01% DMSO. PC: Parental cell. Acta Biochim Biophys Sin (2013) | Page 4
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Casticin decreases invasion of LCSLCs In addition, in view of the important role of CSCs in cancer early metastasis, the Transwell invasion assay was used to
investigate the effects of casticin on invasion of LCSLCs. The results showed that casticin (1, 5, 10 mM) could inhibit the invasion of SFCs from A549 cells in a dose-dependent manner (Fig. 2D,E). It is reported that the MMP-9 is highly expressed in LCSLCs from A549 cells than in parent cells [25]. In this study, the MMP-9 activity in SFCs from A549 cells was found to be decreased gradually with the increase of casticin concentration (Fig. 2F). These data indicate that casticin can suppress the invasion of LCSLCs, possibly via reducing MMP-9 activity.
Casticin suppresses lung cancer stem-like cells
Akt and the expression of stem cell markers CD44, CD133, and ALDH1 in SFCs from A549 cells (Fig. 3D), and significantly decrease the number of SFCs (Fig. 3E). Moreover, LY294002 could augment the down-regulation of p-Akt (Fig. 3F) and the number reduction of SFCs (Fig. 3G) induced by casticin. These results suggest that Akt plays an important role in maintaining stem cell features of LCSLCs, and the mechanisms underlying casticin suppressing LCSLCs are partly via the down-regulation of pAkt.
Discussion We isolated the SFC subpopulation from human NCLSC A549 cells using a sphere-forming enrichment culture method with selective serum-free medium. The results showed that the SFCs could proliferate and form a new sphere, confirming that the SFC subpopulation possessed self-renewal capacity. Then we further discovered that the expression levels of CSC markers CD133, CD44, and ALDH1 in SFCs were much higher than that in parental cells. CD133, CD44, and ALDH1 are important CSC
markers in sorting and identification of various types of CSCs, especially in lung cancer. CD133 often serves as a stemness biomarker for CD133þ CSCs. CD133þ cells are related to the maintenance, metastasis and drug-resistance of lung cancer [9,26,27]. CD133þ cells from primary NSCLC tissue have higher tumorigenic potential in vivo than the CD1332 counterpart [28]. CD44 is also an important stemness biomarker in lung cancer. Recent studies have shown that CD44þ cells from NSCLC have differentiation potential in vivo, express the pluripotency genes OCT4/POU5F1, NANOG, and SOX2, and are more resistant to cisplatin treatment than CD442 cells [29]. It has been demonstrated that the CD133þCD44þ cell subpopulation in A549 cells possesses the continuous proliferative capacity and differentiation potential [30]. ALDH1 has been reported to be over-expressed in NSCLC stem-like cells [12], and the ALDH1A1þ lung cancer cells are more resistant to EGFR tyrosine kinase inhibitor gefitinib than the ALDH1A12 cells [13]. Our results showed that the sorted SFCs from A549 cells expressing higher levels of CD133, CD44, and ALDH1, had Acta Biochim Biophys Sin (2013) | Page 5
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Figure 3. Casticin inhibits the self-renewal of LCSLCs via down-regulation of pAkt Casticin reduced CD133, CD44 and ALDH1 expression in SFC from A549 cells (A). The pAkt was highly expressed in SFC (B). Casticin decreased pAkt level in the SFC (C). Akt inhibitor LY294002 reduced pAkt, CD44, CD133 and ALDH1 expression in SFC (D), and decreased the number of SFC (E). LY294002 augmented down-regulation of pAkt (F) and reduction of the number of SFC (G) induced by casticin. *P , 0.05 vs. 0.01% DMSO or the indicated group.
Casticin suppresses lung cancer stem-like cells
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suggest that casticin is a candidate compound for curing lung cancer via eliminating CSCs.
Acknowledgements We thank Dr. Xiyun Deng from Hunan Normal University for reviewing the manuscript.
Funding This work was supported by the Project of Scientific Research of Hunan Province the Administration Bureau of Traditional Chinese Medicine (201269), the Youth Fund of Hunan Normal University (110637), and the Hunan province Science and Technology Project (2011FJ4144).
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Original Article
Casticin suppresses self-renewal and invasion of lung cancer stem-like cells from A549 cells through down-regulation of pAkt Fei Liu1†, Xiaozheng Cao1†, Zhihong Liu2, Hui Guo2, Kaiqun Ren1, Meifang Quan1, Yuan Zhou1, Honglin Xiang1, and Jianguo Cao1 * 1
College of Medicine, Hunan Normal University, Changsha 410013, China Department of pathology, Hunan Provincial Tumor Hospital, Changsha 410013, China † These authors contributed equally to this work. *Correspondence address. Tel/Fax: þ86-731-88912434; E-mail: [email protected] 2
Keywords lung cancer; casticin; cancer stem cells; pAkt; therapeutic action Received: May 22, 2013
Accepted: September 2, 2013
Introduction Lung cancer is the most common cause of cancer deaths worldwide for its high incidence and mortality. Globally, more than one million new lung cancer cases were reported each year [1,2]. Lung cancer is generally subdivided into
small cell lung cancer and non-small cell lung cancer (NSCLC). The 5-year survival for NSCLC is less than 9% in developing countries [2], and no more than 15% in USA [3]. So it is necessary to explore the novel therapeutic approaches for improving life quality of patients with lung cancer including NSCLC. Recently, more and more researchers are focusing on cancer stem cells (CSCs), trying to find new breakthrough point for curing cancer. The CSCs possess self-renewal capacity and differentiation potential, and can reconstruct the phenotypic and histologic heterogeneity of its parent tumor when transplanted in vivo [4]. The CSC theory presumes that the tumor carcinogenesis, recurrence, and resistance are caused by the existence of a small subpopulation of CSCs. Recent studies are mainly involved in characterizing CSCs from different tumor cell lines or solid tumor tissues including glioblastoma, melanoma, breast cancer, lung cancer, etc., and their crucial signal molecules [5–8]. CSCs can be enriched or identified using cell surface markers via flow cytometry and immunomagnetic beads sorting system [9,10], or sphere-forming assay [4,11] in selective serum-free medium. In NSCLC, CSCs are commonly characterized as a subpopulation of cells expressing CD133 [10], or aldehyde dehydrogenase (ALDH) [12,13] or as a side population [14] of cells capable of effluxing Hoechst dye. Identification of CSCs in tumor cells is important, but studies on this area are just the beginning. What is more important is to find the effective therapeutic strategies, including screening of novel drugs targeting CSCs. Casticin (5,30 -dihydroxy-3,6,7,40 -tetramethoxyflavone) is a main active ingredient of Fructus Viticis Simplicifoliae, which is often used as the traditional Chinese medicine for the treatment of certain types of cancer. Studies have shown that casticin possesses extensive anticancer bioactivity for cancers including lung cancer [15], cervical cancer [16], leukemia [17,18], colon cancer [19], hepatocellular carcinoma Acta Biochim Biophys Sin (2013) | Page 1
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A subpopulation of cancer stem cells is recognized as the cause of tumorigenesis and spreading. To investigate the effects of casticin (5,30 -dihydroxy-3,6,7,40 -tetramethoxyflavone), derived from Fructus Viticis Simplicifoliae, on lung cancer stem cells, we isolated and identified a subpopulation of lung cancer stem-like cells (LCSLCs) from non-small-cell lung carcinoma A549 cells with the features including selfrenewal capacity and high invasiveness in vitro, elevated tumorigenic activity in vivo, and high expression of stemness markers CD133, CD44, and aldehyde dehydrogenase 1 (ALDH1), using serum-free suspension sphere-forming culture method. We then found that casticin could suppress the proliferation of LCSLCs in a concentration-dependent manner with an IC50 value of 0.4 mmol/L, being much stronger than that in parental A549 cells. In addition, casticin could suppress the self-renewal and invasion of LCSLCs concomitant with decreased CD133, CD44, and ALDH1 protein expression and reduced MMP-9 activity. Further experiments showed that casticin suppressed selfrenewal and invasion at least partly through down-regulation of Akt phosphorylation. In conclusion, casticin suppressed the characteristics of LCSLCs, suggesting that casticin may be a candidate compound for curing lung cancer via eliminating cancer stem cells.
Casticin suppresses lung cancer stem-like cells
cells [20], and so on [21,22]. We have found that casticin could inhibit self-renewal of glioma stem-like cells [23]. However, the effects of casticin on lung cancer stem-like cells (LCSLCs) and its mechanisms remain elusive. In this study, we enriched and identified LCSLCs from NSCLC A549 cells, and found that casticin could suppress the self-renewal and invasion of LCSLCs, accompanied with a reduced CD133, CD44, and ALDH expression. The underlying mechanisms were associated with the decrease of phosphorylation Akt, which was reported to play a crucial role in the maintenance of CSC features.
Materials and Methods
Matrigel invasion assay Two thousands cells incubated in serum-free medium were added on the top of the transwell chamber coated with fresh Matrigel (Gibco). The medium supplemented with 10% FBS, as the chemical inducing agent, was added to the bottom of the transwell chamber. After incubation for 48 h, the cells on top of the chamber were scraped off using a cotton swab. And the cells in the bottom of the chamber were fixed with methanol and stained with Wright staining solution. The cells invading through the membrane were counted under an optical microscope.
Sphere-forming and self-renewal assay To obtain sphere cultures, cells were plated at a density of 5 103 cells/well in 6-well ultra-low plates (Corning, Acton, USA) containing serum-free medium DMEM/F12 (Gibco, Carlsbad, USA), supplemented with commercial hormone mix B27 (Gibco), 20 ng/ml EGF (PeproTech, Rocky Hill, USA), 10 ng/ml bFGF (PeproTech), 0.4% bovine serum albumin (Gibco), 4 mg/ml insulin (Gibco), 100 U/ml penicillin and 100 U/ml streptomycin at 378C. After being cultured for 6 days, the lung cancer spheres were collected, dissociated into single cell suspension and resuspended in fresh medium for serial subcultivation every 6 days. To investigate the self-renewal capacity of lung cancer sphere-forming cells (SFCs), single cell suspension prepared from the lung cancer sphere was diluted to 500 cells/ml. Two microliters of the single cell suspension was plated in 96-well ultra-low plates containing 150 ml serum-free medium per well. Wells containing no cells or more than one cell were excluded, and those with one cell were marked and monitored daily under a microscope (Nikon Eclipse TE2000-S, Nikon, Japan) for 6 days and the colonies were counted. To investigate the effects of casticin on self-renewal of SFCs, single cell suspension of SFCs was plated at a density of 2 103 cells/ml in 6-well ultra-low plates. Different concentrations of casticin (1.0, 5.0, and 10.0 mM) were added to medium. After being cultured for 6 days, the colonies were counted under a microscope.
MMP-9 activity assay The SFCs were plated at a density of 1 106 cells/well in 6-well ultra-low plates containing serum-free medium. After 24 h incubation, the medium was replaced with serum-free medium containing different concentrations of casticin (1.0, 5.0, 10.0 mM), cultured for additional 72 h. Then the medium was collected and subject to MMP-9 activity assay using human active MMP-9 fluorokine E kit (R&D Systems, Minneapolis, USA) according to manufacturer’s instructions. The activity unit was defined as ng/ml/106 cells.
MTT [3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay The SFCs or parental cells were plated at 5000 cells/well in 96-well ultra-low plates. The cells were first incubated with
Western blot analysis The cells were lysed using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2 mM EDTA, 0.2% NP-40, 10% glycerol, 1 M b-Me, 1 mg/ml aprotinin, 0.5 mg/ml aprotinin
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Tumorigenicity assay The SFCs or parental cells were injected subcutaneously into the back of 4-week-old BALB/C-nude mice (supplied by the Shanghai Experimental Animal Center, Chinese Academy of Sciences, Shanghai, China). The mice were reared for 2 months, and the tumor growth and tumorigenic time were examined visually. Tumor volumes were calculated in accordance with the formula: V (transplanted tumor volume, mm3) ¼ L (longest diameter, mm) W (minimum diameter, mm)2 0.5. At the end of experiment, the mice were sacrificed under deep anesthesia with pentobarbital. The tumors were then dissected, weighed and fixed with 10% neutral formalin. Paraffin sections were prepared following routine procedures for H&E staining.
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Cell culture Human lung cancer A549 cell line was purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China), cultured in RPMI-1640 (HyClone, Logan, USA), supplemented with 10% fetal bovine serum (FBS) at 378C in 5% CO2.
casticin at the indicated concentrations for 48 h, and then incubated with 5 mg/ml MTT for 4 h. Lastly, 100 ml of dimethyl sulfoxide (DMSO) was added to each well, and the absorbance at 570 nm (A570) each well was detected on a plate reader (Bio-Tek EXL-800, Winooski, USA). The relative cell proliferation inhibition rate ¼ (1— experimental group A570 mean/control group A570 mean) 100%. IC50 (50% inhibiting concentration) was calculated by Prism (GraphPad Software, San Diego, USA).
Casticin suppresses lung cancer stem-like cells
Statistical analysis Data were presented as the mean + standard deviation. To assess the statistical significance of differences, student’s t test was performed. P , 0.05 was regarded as statistically significant.
Results Lung cancer SFCs derived from A549 cell lines have the self-renewal capability In order to enrich LCSLCs from human lung cancer A549 cell line, stem cell conditioned medium suspension culture method was used. The results showed that A549 cells could form non-adherent sphere which is called lung cancer SFCs (Fig. 1A2). The results suggested that LCSLCs exist in human lung cancer A549 cells. We next performed the SFCs subculture and tumor sphere formation test. It was found that the single cell of SFC from lung cancer A549 was able to form a new sphere (Fig. 1B), suggesting that the SFCs from A549 cells have the ability of self-renewal. Lung cancer SFCs overexpress stem cell markers Furthermore, we investigated the expressions of CSC biomarker CD44, CD133, and aldehyde dehydrogenase 1 (ALDH1) in SFCs using western blotting. The results demonstrated that, compared with human lung cancer parental cells, the CD44, CD133, and ALDH1 protein expression levels were apparently increased in their SFCs (Fig. 1C). Lung cancer SFCs possess higher tumorigenicity In addition, the xenograft tumor model in nude mouse was used to investigate the tumorigenicity of A549 cells and
Figure 1. Identification of LCSLCs from lung cancer A549 cells Parental cell A549 (A1) and its SFC enriched by stem cell conditioned medium suspension culture method (A2). The single cell of SFC formed a new sphere (B). The CD44, CD133 and ALDH1 protein expression levels in SFC and their PCs (C). The tumorigenicity (D) and immunohistochemical characteristics (E) of A549 cells and its SFC xenograft tumor in Balb/c-nu immunodeficient mice. PC: Parental cell.
their SFCs in Balb/c-nu immunodeficient mice. The results showed that 1 104 A549 SFCs were enough to form tumors in vivo (Fig. 1D), and the tumors have similar immunohistochemical characteristics with its parental cell xenograft tumor (Fig. 1E). However, in the same model, at least 1 106 A549 parental cells were required to induce stable tumor formation (Table 1). Our data indicate that the SFCs from A549 cell line have higher tumorigenicity in vivo than parental cells. The above data confirmed that SFCs from human lung cancer A549 cell line cells possessed characteristics of CSC. Therefore, we can, at least, deduce that the lung cancer SFCs prepared by us belong to LCSLCs, and the LCSCs were possibly enriched in them.
Casticin inhibits the self-renewal capability of LCSLCs The unlimited proliferation potential is an important feature of CSCs [24]. Genistein is a natural polyphenolic compound that can preferentially inhibit the proliferative activity of the pancreatic CSCs than parent cells [23]. Our results Acta Biochim Biophys Sin (2013) | Page 3
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aldehyde peptide, 0.1 mM Na3VO4, 0.5 mM 4 NPP, 0.5 mM NaF, and protease inhibitors), and the total cell extracts were prepared. The protein content was measured using the Bradford method. The proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride (PVDF) membrane. After being blocked with 5% non-fat milk in phosphate buffered saline containing Tween-20 (PBST), the PVDF membrane was incubated using the following mouse anti-human primary monoclonal antibodies: anti-pAkt (Ser473) (1 : 500; Cell signaling, Beverly, USA), anti-Akt (1 : 2000; cell signaling), anti-CD44 (1 : 500; cell signaling), anti-CD133 (1 : 500; cell signaling), antiALDH1 (1 : 500; cell signaling), and anti-b-actin (1 : 200; Sigma, St. Louis, USA), with slight shaking at 48C overnight. Then the membrane was incubated with peroxidase-labeled secondary antibody for 1 h, and the signal was detected using an enhanced chemiluminescence kit (Amersham Biosciences, Buckinghamshire, UK) according to manufactures’ protocols.
Casticin suppresses lung cancer stem-like cells
showed that casticin (0.1, 1, 3, 10, 30 mM) could inhibit the proliferation of human lung cancer SFCs in a dosedependent manner (Fig. 2A), and the IC50 of casticin on A549 cells and their SFCs were 14.3 and 0.4 mmol/L, respectively, suggesting that casticin is able to preferentially suppress the proliferation activity of LCSLCs. Furthermore, casticin (1, 5, 10 mM) could reduce the size and number of SFCs from A549 cells in a dose-dependent manner (Fig. 2B,C), suggesting that casticin can suppress the self-renewal of LCSLCs.
Table 1. Xenotransplantation of human lung cancer A549 cells and its SFCs into Balb/c-nu immunodeficient mice
Cell
Inoculum size Tumor incidence Latency period (d)
A549 cells 1 104 1 105 1 106 SFCs 2 103 1 104 1 105
0/4 0/4 2/4 2/4 4/4 4/4
– – 31 29 19 11
Casticin reduces CD133, CD44, and ALDH1 expression in LCSLCs Western blot analysis showed that casticin (1, 5, 10 mM) reduced CD133, CD44, and ALDH1 protein expression in SFC from A549 cells, in a dose-dependent manner (Fig. 3A). It suggests that casticin can decrease CSC marker expression in LCSLCs. Casticin inhibits the self-renewal of LCSLCs via down-regulation of pAkt Western blot analysis showed that the phosphorylation levels of Akt in SFCs from A549 cells were higher than those in A549 cells (Fig. 3B). Casticin could decrease phosphorylated Akt ( p-Akt) expression in the SFCs in a dosedependent manner (Fig. 3C). Akt inhibitor LY294002 (5, 10, 20 mM) could effectively reduce phosphorylation of
Figure 2. Effects of casticin on LCSLCs Casticin inhibited proliferation (A) and reduced the size (B) and number (C) of SFC from A549 cells. Casticin suppressed the invasion of SFC from A549 cells (D) and (E). Casticin decreased MMP-9 activity (F). *P , 0.05 vs. 0.01% DMSO. PC: Parental cell. Acta Biochim Biophys Sin (2013) | Page 4
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Casticin decreases invasion of LCSLCs In addition, in view of the important role of CSCs in cancer early metastasis, the Transwell invasion assay was used to
investigate the effects of casticin on invasion of LCSLCs. The results showed that casticin (1, 5, 10 mM) could inhibit the invasion of SFCs from A549 cells in a dose-dependent manner (Fig. 2D,E). It is reported that the MMP-9 is highly expressed in LCSLCs from A549 cells than in parent cells [25]. In this study, the MMP-9 activity in SFCs from A549 cells was found to be decreased gradually with the increase of casticin concentration (Fig. 2F). These data indicate that casticin can suppress the invasion of LCSLCs, possibly via reducing MMP-9 activity.
Casticin suppresses lung cancer stem-like cells
Akt and the expression of stem cell markers CD44, CD133, and ALDH1 in SFCs from A549 cells (Fig. 3D), and significantly decrease the number of SFCs (Fig. 3E). Moreover, LY294002 could augment the down-regulation of p-Akt (Fig. 3F) and the number reduction of SFCs (Fig. 3G) induced by casticin. These results suggest that Akt plays an important role in maintaining stem cell features of LCSLCs, and the mechanisms underlying casticin suppressing LCSLCs are partly via the down-regulation of pAkt.
Discussion We isolated the SFC subpopulation from human NCLSC A549 cells using a sphere-forming enrichment culture method with selective serum-free medium. The results showed that the SFCs could proliferate and form a new sphere, confirming that the SFC subpopulation possessed self-renewal capacity. Then we further discovered that the expression levels of CSC markers CD133, CD44, and ALDH1 in SFCs were much higher than that in parental cells. CD133, CD44, and ALDH1 are important CSC
markers in sorting and identification of various types of CSCs, especially in lung cancer. CD133 often serves as a stemness biomarker for CD133þ CSCs. CD133þ cells are related to the maintenance, metastasis and drug-resistance of lung cancer [9,26,27]. CD133þ cells from primary NSCLC tissue have higher tumorigenic potential in vivo than the CD1332 counterpart [28]. CD44 is also an important stemness biomarker in lung cancer. Recent studies have shown that CD44þ cells from NSCLC have differentiation potential in vivo, express the pluripotency genes OCT4/POU5F1, NANOG, and SOX2, and are more resistant to cisplatin treatment than CD442 cells [29]. It has been demonstrated that the CD133þCD44þ cell subpopulation in A549 cells possesses the continuous proliferative capacity and differentiation potential [30]. ALDH1 has been reported to be over-expressed in NSCLC stem-like cells [12], and the ALDH1A1þ lung cancer cells are more resistant to EGFR tyrosine kinase inhibitor gefitinib than the ALDH1A12 cells [13]. Our results showed that the sorted SFCs from A549 cells expressing higher levels of CD133, CD44, and ALDH1, had Acta Biochim Biophys Sin (2013) | Page 5
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Figure 3. Casticin inhibits the self-renewal of LCSLCs via down-regulation of pAkt Casticin reduced CD133, CD44 and ALDH1 expression in SFC from A549 cells (A). The pAkt was highly expressed in SFC (B). Casticin decreased pAkt level in the SFC (C). Akt inhibitor LY294002 reduced pAkt, CD44, CD133 and ALDH1 expression in SFC (D), and decreased the number of SFC (E). LY294002 augmented down-regulation of pAkt (F) and reduction of the number of SFC (G) induced by casticin. *P , 0.05 vs. 0.01% DMSO or the indicated group.
Casticin suppresses lung cancer stem-like cells
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suggest that casticin is a candidate compound for curing lung cancer via eliminating CSCs.
Acknowledgements We thank Dr. Xiyun Deng from Hunan Normal University for reviewing the manuscript.
Funding This work was supported by the Project of Scientific Research of Hunan Province the Administration Bureau of Traditional Chinese Medicine (201269), the Youth Fund of Hunan Normal University (110637), and the Hunan province Science and Technology Project (2011FJ4144).
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