Anlotinib exerts anti‐cancer efficiency on lung cancer stem cells in vitro and in vivo through reducing NF‐κB activity

Abstract Anlotinib is a multi‐target tyrosine kinase inhibitor. Previous studies confirmed that anlotinib exerts anti‐cancer efficiency. However, the functional roles of anlotinib on cancer stem cells (CSCs) are yet to be elucidated. In this study, lung CSCs were isolated and identified in vitro, and mouse xenografts were established in vivo. MTT assays, tumour sphere formation assays, TdT‐mediated dUTP nick‐end labelling (TUNEL) staining, Annexin V‐FITC/PI staining, immunofluorescence analysis and Western blot were performed to investigate the anti‐cancer effects of anlotinib on lung CSCs. The results showed that anlotinib inhibits the growth of lung CSCs in vitro and in vivo. In addition, anlotinib induced apoptosis of these cells along with down‐regulated expression level of Bcl‐2 whereas up‐regulated Bax and cleaved caspase‐3 expression. It also sensitized lung CSCs to the cytotoxicity of cisplatin and paclitaxel; the tumour sphere formation and expression levels of multiple stemness‐associated markers, such as ALDH1 and CD133, were also decreased. Furthermore, the underlying mechanism indicated that anlotinib reduces the phosphorylated levels of NF‐κB p65 and IκB‐α in lung CSCs. Taken together, these findings suggested that anlotinib exerts potent anti‐cancer effects against lung CSCs through apoptotic induction and stemness phenotypic attenuation. The mechanism could be associated with the suppression of NF‐κB activity.

resistance to chemotherapy and high expression levels of stemnessassociated markers, such as ALDH1, CD133, Sox2 and Oct4, might be responsible for tumour recurrence and metastasis in cancer patients irrespective of comprehensive anti-cancer therapies, including chemoradiotherapy, targeted therapy and immunotherapy. [4][5][6] In the lung cancer cell lines or lung cancer patients, CSCs were isolated and involved in drug resistance and tumour recurrence. 7,8 Therefore, developing new anti-cancer agents targeting lung CSCs (LCSCs) and exploring the molecular mechanisms underlying these processes are promising.
Anlotinib is a tyrosine kinase inhibitor targeting VEGFR, PDGFR, FGFR and c-Kit. 9 Previous studies reported that anlotinib exerts potent anti-cancer efficiency against several types of cancer, including thyroid cancer, osteosarcoma and renal cell carcinoma. [10][11][12] Recently, Hu et al clarified that anlotinib inhibits proliferation and induces apoptosis of KRAS-mutated lung cancer cells. 13 However, the anti-cancer activities of anlotinib against LCSCs and the underlying molecular mechanism have not yet been elucidated.
In this study, we aimed to assess the potential functional roles of anlotinib on LCSCs in vitro and in vivo and explore the molecular mechanism involved in this process.

| Cell lines and culture
The human lung adenocarcinoma cell lines, PC-9 and HCC827, purchased from Shanghai Cell Biology Institute of Chinese Academy of Sciences (Shanghai, China), were cultured in RPMI 1640 containing 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) in a humidified environment with 5% CO 2 at 37°C.
The medium was changed every other day until tumour sphere formation within 5-7 days. Passage 9 of PC-9-and HCC827-derived LCSCs were used to identify the stemness phenotype, including drug resistance, tumour sphere formation and the expression levels of stemness-associated markers. Subsequently, passage 10 of PC-9-and HCC827-derived LCSCs were used in the subsequent experiments.

| Annexin V-FITC/PI double staining
Apoptosis was assessed using an Annexin V-FITC/PI apoptotic detection kit (Keygen, Biotech), according to the manufacturer's instructions. Briefly, PC-9 cells, HCC827 cells, PC-9-derived LCSCs or HCC827-derived LCSCs, administered anlotinib (5, 10 or 20 μM) for 3 days, were incubated with FITC-conjugated Annexin V and PI at room temperature for 15 minutes away from light. Then, these cells were subjected to flow cytometry (BD FACSCalibur; BD Bioscience) and sorted into intact (Annexin V and PI double-negative), early apoptotic (Annexin V-positive), late apoptotic (Annexin V and PI double-positive) and necrotic (PI-positive) cells.

| Animals and ethics statement
All studies were approved by the Institutional Animal Care and Use Committee of Sichuan University (SCU). All animal feeding and experiments were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee at SCU.
Five-week-old female nude mice were purchased from the Experimental Animal Center, Sichuan University. Each animal was inoculated with 1 × 10 6 PC-9-derived LCSCs subcutaneously in the right flank. After allowed to grow for 1 week to reach a tumour volume of approximately 50-100 mm 3 , the animals were randomly divided into three groups (n = 6). The treatment group animals were treated with anlotinib at the concentrations of 10 and 20 mg/kg, and the control group animals were treated with normal saline intraperitoneally once daily for 4 weeks. Throughout the experiment, the tumour volumes were assessed by measuring the respective lengths and widths with vernier callipers.
The tumour volumes were derived using the standard formula: V = width 2 × length × 0.5. At the end of the treatment, the animals were euthanized by carbon dioxide asphyxiation, and tumour tissues were extracted, weighed and processed for fluorescence staining and Western blot analysis.

| TUNEL staining
The apoptosis detection in tumour tissues was carried out

| Immunofluorescence staining
PC-9-or HCC827-derived LCSCs were permeabilized in 0.2% Triton X-100 for 10 minutes at room temperature and then blocked with 5% BSA for 30 minutes. Then, the cells were incubated with primary antibody NF-κB p65 (1:500; Cell Signaling Technology (CST)) at 4℃ overnight, followed by secondary antibody (ZSGB-BIO) for 1 hour at 37°C for immunofluorescence staining. Finally, the cell nuclei were stained by DAPI for 10 minutes, and images were captured under a fluorescence microscope.
Frozen sections (5 μm) of tumour tissues from animal experiments mentioned above were assessed for ALDH1 and CD133 expression. The sections were fixed in 4% paraformaldehyde, blocked with 5% BSA for 30 minutes and incubated at 4°C overnight with the following primary antibodies: ALDH1 and CD133 (1:1000, Abcam).
Then, the cells were incubated with appropriate secondary antibodies for 1 hour at 37°C. DAPI was used to visualize nuclei. The images were captured under a fluorescence microscope linked to a camera, and the fluorescence intensities were measured using the Image J software (NIH).

| Statistical analysis
Data were analysed by GraphPad Version 6.0 and expressed as mean ± standard deviation (SD). Significant differences between F I G U R E 2 Anlotinib inhibits proliferation and induces apoptosis of PC-9-and HCC827-derived LCSCs. A, Cell viability of PC-9 cells, PC-9-derived LCSCs, HCC827 cells and HCC827-derived LCSCs after treatment with various concentrations of anlotinib. PC-9 vs. PC-9derived LCSCs; HCC827 vs. HCC827-derived LCSCs. B, Annexin V-FITC/PI double staining was used for apoptosis detection in PC-9 vs. PC-9-derived LCSCs; HCC827 vs. HCC827-derived LCSCs after treatment with various concentrations of anlotinib. Early and late apoptotic cells were assessed on flow cytometer; the histograms represent the proportions of early, late and total apoptotic cells, respectively. C, Western blot analysis of Bcl-2, Bax and cleaved caspase-3 in PC-9 cells, PC-9-derived LCSCs, HCC827 cells and HCC827-derived LCSCs after treatment with various concentrations of anlotinib. Con vs. 5, 10, or 20 μM. * P < 0.05; ** P < 0.01. Data are expressed as mean ± SD of three independent experiments performed in triplicate | 5553 LI et aL. groups were analysed using Student's t test and one-or two-way analysis of variance (ANOVA). Statistical significance was defined as P < 0.05 for all tests.

| Identification of stemness phenotypic characteristics in PC-9-and HCC827-derived LCSCs
As shown in Figure 1A, PC-9-and HCC827-derived LCSCs were grown as suspension cultures and exhibited a sphere-like morphology, which was remarkably different from parental PC-9 cells and HCC827 cells, respectively. In addition, the sphere formation assay revealed that along with increasing passages of PC-9-and HCC827-derived LCSCs, the SFE of these cells was enhanced gradually compared with the SFE in parental PC-9 cells and HCC827 cells, respectively ( Figure 1B). Moreover, the PC-9-and HCC827-derived respectively, and both were much higher than that obtained for HCC827 cells (3.96 ± 0.72 µM and 7.73 ± 0.32 nM) ( Figure 1C).

| Anlotinib attenuates stemness phenotypic features of PC-9-and HCC827-derived LCSCs
To investigate the effects of anlotinib on the stemness phenotype in PC-9-and HCC827-derived LCSCs, we assessed the drug resistance, SFE and the changes in the expression levels of stemness-associated markers in these cells. As shown in Figure 3A

| Anlotinib exerts anti-cancer effects in vivo
As demonstrated above, anlotinib induced apoptosis and attenuated the stemness phenotype of PC-9-and HCC827-derived LCSCs in vitro. However, whether anlotinib exerted anti-cancer properties against LCSCs in vivo is yet unclear. To further assess the functional roles of anlotinib, PC-9-derived LCSC tumourbearing mice were established and intraperitoneally administered 10 mg/kg and 20 mg/kg anlotinib, respectively, for 4 weeks. As shown in Figure 4A, anlotinib significantly inhibited the tumour growth of PC-9-derived LCSC tumour-bearing mice. In addition, F I G U R E 3 Anlotinib attenuates stemness phenotype of PC-9-and HCC827-derived LCSCs. A, Cell viability of anlotinib (20 μM) pretreated PC-9-and HCC827-derived LCSCs after administration of various concentrations of paclitaxel and cisplatin, respectively. Pre-treated PC-9-derived LCSCs vs. PC-9-derived LCSCs; Pre-treated HCC827-derived LCSCs vs. HCC827-derived LCSCs. B, Histograms show tumour sphere formation efficiency of PC-9-and HCC827-derived LCSCs after treatment with various concentrations of anlotinib. C, Western blot analysis of ALDH1, CD133, Oct4 and Sox2 in PC-9-and HCC827-derived LCSCs after treatment with various concentrations of anlotinib. Con vs. 10 or 20 μM. * P < 0.05; ** P < 0.01. Data are expressed as mean ± SD of three independent experiments performed in triplicate | 5555

LI et aL.
TUNEL staining in xenografts demonstrated that anlotinib promoted apoptosis of PC-9-derived LCSCs in vivo along with decreased Bcl-2 expression and increased levels of Bax and cleaved caspase-3 ( Figure 4B,D). Furthermore, immunofluorescence staining of ALDH1 and CD133 in xenografts verified that the fluorescence intensity of these stemness-associated markers was decreased after administration of anlotinib in PC-9-derived LCSC tumour-bearing mice. These findings were further verified by Western blot analysis in xenografts ( Figure 4C,D).

| Anlotinib suppresses NF-κB activity in PC-9-and HCC827-derived LCSCs
Previous reports indicated that the regulation of NF-κB pathway was closely associated with apoptosis and stemness phenotype maintained in CSCs. [15][16][17][18] To clarify the effects of anlotinib on apoptosis and stemness phenotype attenuation in PC-9-and HCC827derived LCSCs involved with the regulation of NF-κB activity, we examined the changes in the NF-κB pathway protein expression post-anlotinib treatment. As shown in Figure 5A, the expression level of cytoplasmic NF-κB p65 was up-regulated, whereas the nuclear level was down-regulated. Strikingly, the phosphorylated levels of NF-κB p65 in both cytoplasm and nuclei were reduced after anlotinib treatment. On the other hand, anlotinib treatment decreased the phosphorylated level of IκBα in PC-9-and HCC827-derived LCSCs. Fluorescence staining of NF-κB p65 further verified that anlotinib blocked the translocation of NF-κB p65 to the nucleus in PC-9-and HCC827-derived LCSCs ( Figure 5B).
Together, these results suggested that anlotinib suppressed NF-κB activity in PC-9-and HCC827-derived LCSCs. In order to further explore whether the regulation of NF-κB pathway is related to apoptosis induction and stemness phenotype attenuation in these cells, BAY11-7082 (Beyotime Institute of Biotechnology), a specific NF-κB inhibitor, was utilized. As shown in Figure 5C, sphere formation assay demonstrated that BAY11-7082 decreased the SFE of PC-9-and HCC827-derived LCSCs. In addition, the expression level of stemness-associated markers (ALDH1 and CD133) was reduced after BAY11-7082 treatment ( Figure 5D). We also observed that in BAY11-7082 treated PC-9-and HCC827-derived LCSCs, the level of anti-apoptotic Bcl-2 protein was down-regulated, whereas that of pro-apoptotic proteins Bax and cleaved caspase-3 was upregulated in a dose-dependent manner ( Figure 5D). These data indicated that anlotinib-induced apoptosis and anlotinib -attenuated stemness phenotype of PC-9-and HCC827-derived LCSCs might at least partially, be attributed to the suppression of NF-κB activity.

| D ISCUSS I ON
Lung cancer is one of the most lethal malignancies, and the 5-year survival rate is <18%. Despite improvements in the present therapies of lung cancer, including the expansion of indications for surgical resection, modification of chemotherapeutic drugs, accurate radiotherapy, continuous development of targeted drugs and burgeoning immunotherapy, the anti-cancer efficiency is still limited.
Advanced lung cancer patients experience tumour recurrence and metastasis after a series of therapeutic methods against cancer.
Therefore, exploring the pathogenesis of lung cancer and developing new targeted agents is an urgent requirement.
CSCs, also known as tumour-initiating cells, constitute a small heterogeneous cell population within many types of cancers, including lung, breast, brain, pancreas and colon. 19 Though the proportion of CSCs is low, approximately <6%-7%, the cells are endowed with stemness phenotype characteristics, including enhanced selfrenewal ability, resistance to chemotherapy and high expression levels of stemness markers, such as ALDH1, Sox2, Oct4 and CD133. 20 Current evidence suggests that the presence of CSCs drives the metastasis and recurrence of tumour; thus, eliminating CSCs might be a promising strategy combined with chemotherapy or other anticancer therapies. 21 Together, the establishment of the CSC model is imperative. In this study, we utilized the non-adhesive culture system and isolated and identified the stemness phenotype of lung CSCs from parental PC-9 cells and HCC827 cells.
CSCs are characterized by self-renewal ability to maintain their proportion in tumours and the potential to differentiate into non-tumorigenic bulk tumour cells; the self-renewal is tightly associated with the pathological ability to regenerate tumours. 15 The sphere formation assay is an in vitro method commonly used as a functional reporter of the self-renewal ability of CSCs. 22,23 Yang et al reported that the SFE in primary and secondary A549dervied LCSCs was much higher than that in parental A549 cells. 24 In addition, Li et al verified that cervical cancer cell-derived CSCs exhibited increased SFE compared with parental cervical cancer cells, and the efficiency of sphere formation in these CSCs stabilized after a series of passages. 14 In the present study, the sphere formation assay demonstrated that the SFE of PC-9-and HCC827-derived LCSCs was increased gradually and also tended to be stable after experiencing multiple and serial propagations of the spheres compared with the parental PC-9 cells and HCC827 cells, respectively. Resistance to the cytotoxicity of chemotherapeutic agents is an important trait of CSCs. Kanwal et al demonstrated that CD133-positive prostate cancer cells were endowed with stem cell-like characteristics, including therapeutic F I G U R E 4 Anti-cancer effects of anlotinib in PC-9-derived LCSC tumour-bearing mice. A, Tumour volumes and weights were measured after treatment with anlotinib. B, TNUEL staining of xenografts after treatment with anlotinib. C, Immunofluorescence staining of CD133 and ALDH1 in xenografts after treatment with anlotinib; histograms show fluorescence intensity of CD133 and ALDH1. D, Western blot analysis of Bcl-2, Bax, cleaved caspase-3, CD133 and ALDH1 in xenografts after treatment with anlotinib. Con vs. 10 or 20 mg/kg. * P < 0.05; ** P < 0.01; *** P < 0.001. Data are expressed as mean ± SD of three independent experiments performed in triplicate | 5557 LI et aL. resistance to docetaxel, which is a chemotherapeutic drug acting efficiently on CD133-negative prostate cancer cells. 25 In addition, He et al demonstrated that breast cancer cell line BT549-derived CSCs were resistant to the cytotoxicity of doxorubicin, commonly used as a systemic therapeutic drug for breast cancer patients. 26 In this study, we also verified that PC-9-and HCC827-derived LCSCs were resistant to cisplatin and paclitaxel, indicating that CSCs might be responsible for the drug resistance cancer patients despite multiline chemotherapy. The stemness-associated markers were previously reported to maintain the phenotypic features in CSCs, and to decrease or to increase the expression levels of these markers could influence the self-renewal ability, chemotherapy resistance or metastatic potential of these cells. Recently, Zhan et al reported that overexpressing Sox2 promoted the stemness phenotypic characteristics, such as colony formation and metastatic ability in bladder cancer cells. 27 Li et al also indicated that silencing of ALDH1 expression through siRNA technology decreased the tumorigenicity and migration ability of breast CSCs. 28 Moreover, Chen et al revealed that knockdown of Oct4 expression in lung CSCs significantly inhibited the abilities of tumour invasion and colony formation. 29 CD133 is a common and vital stemness marker and identified in many types of CSCs, including lung, breast and prostate CSCs, as well as glioma stem cells. [30][31][32] Ding et al verified that CD133 is a liver CSC marker, and reducing CD133 expression in these cells attenuated chemotherapeutic drug 5-Fu resistance in hepatocellular carcinoma. 33 In another study, Lan et al clarified that down-regulated CD133 expression in CD133-positive liver CSCs inhibits the tumour sphere formation, colony formation, and in vivo tumour growth while enhancing the sensitivity of liver CSCs to chemotherapy and radiotherapy. 34 In the present study, we demonstrated that the expression level of stemness-associated markers, including ALDH1, Sox2, Oct4 and CD133, was increased in PC-9-derived LCSCs compared with PC-9 cells, which was consistent with increased stemness phenotype in these cells. However, in HCC827-derived LCSCs, the expression level of Sox2 was low and similar to the parental HCC827 cells, suggesting that Sox2 might not be closely involved with the functional regulation of stemness phenotypic characteristics in HCC827 cells.
These results above indicated that our enriched PC-9-and HCC827-derived LCSCs were featured with stemness phenotype and could be used as CSC models for further investigation.
Anlotinib is a novel tyrosine kinase inhibitor (TKI) approved for advanced lung cancer patients due to obvious antiangiogenic effects in tumour tissues. Typically, anlotinib exerts anti-cancer effects in several types of cancer cells. However, the effect of anlotinib on CSCs and the underlying molecular mechanisms remains unclear. In the present study, we first demonstrated that anlotinib inhibited the proliferation of PC-9-and HCC827-derived LCSCs in vitro.
Apoptosis is a critical mechanism of anti-cancer drugs and is involved with cell proliferation inhibition. A previous study indicated that anlotinib induced apoptosis of KRAS mutant A549 cells. In this study, flow cytometry analysis clarified that anlotinib also induced apoptosis in PC-9-and HCC827-derived LCSCs. Apoptosis is essentially regulated by two large families of proteins: caspase and Bcl-2. 35 Bcl-2 protects the cells from apoptosis, whereas Bax reverses the effect. The caspases are a family of protease enzymes that are mainly classified into initiators (caspase-9) and effectors (caspase-3).
Based on these findings, we assessed the changes in the expression of apoptotic proteins in these cells after anlotinib treatment, and the results showed that the expression levels of Bax and cleaved caspase-3 in PC-9-and HCC827-derived LCSCs were increased, whereas that of Bcl-2 was decreased. In the in vivo assay, we established PC-9-derived LCSC tumour-bearing mice and found that anlotinib suppressed tumour growth. Furthermore, TUNEL staining revealed that the proportion of TUNEL-positive cells in xenografts of PC-9-derived LCSC tumour-bearing mice was increased after anlotinib treatment in addition to the up-regulated expression of Bax and cleaved caspase-3 and the down-regulated expression of Bcl-2.
Next, we assessed the changes in stemness phenotypic characteristics in PC-9-and HCC827-derived LCSCs after anlotinib treatment. The results firstly verified that anlotinib decreased tumour sphere formation and chemotherapy resistance of these LCSCs in vitro. On the other hand, after treatment with anlotinib, the expression level of stemness-associated markers, ALDH1 and CD133, was obviously reduced in PC-9-and HCC827-derived LCSCs as well as the xenografts of PC-9-derived LCSC tumour-bearing mice.
Previous reports suggested that NF-κB pathway was closely involved with apoptosis and the stemness phenotype of CSCs.
Erdogan et al demonstrated that in prostate CSCs, apigenin induced extrinsic caspase-dependent apoptosis in these cells by decreasing the expression level of NF-κB p105 and NF-κB p50. 15 In leukaemia stem cells, Xu et al revealed that disulfiram combined with copper exerted pro-apoptotic effects of these cells via suppression of NF-κB p65 expression. 16 Moreover, Zhang et al suggested that in pancreatic cancer, gemcitabine treatment activated NF-κB signalling pathway by increasing the nuclear translocation of NF-κB p65 and then promoted stemness phenotypic characteristics, such as enhanced sphere formation, migration and chemoresistance as well F I G U R E 5 Anlotinib suppresses NF-κB activity in PC-9-and HCC827-derived LCSCs. A, Western blot analysis of pho-NF-κB p65, NF-κB p65, IκBα and pho-IκBα in PC-9-and HCC827-derived LCSCs after treatment with various concentrations of anlotinib. Con vs. 10, 15, or 20 μM. * P < 0.05; ** P < 0.01; *** P < 0.001. Data are expressed as mean ± SD of three independent experiments performed in triplicate. B, Immunofluorescence staining of NF-κB p65; the cells with nuclear translocation of NF-κB p65 are indicated with red arrows; the scale bar = 100 μm. C, Histograms show tumour sphere formation efficiency of PC-9-and HCC827-derived LCSCs after treatment with various concentrations of BAY11-7082. D, Western blot analysis of Bcl-2, cleaved caspase-3, Bax, ALDH1 and CD133 in PC-9-and HCC827-derived LCSCs after treatment with various concentrations of BAY11-7082. Con vs. 1.25 or 2.5 μM. * P < 0.05; ** P < 0.01. Data are expressed as mean ± SD of three independent experiments performed in triplicate as the expression of stemness markers, including Sox2, Nanog and Bmi1. 17 Similarly, ovarian CSCs presented high expression of NF-κB pathway-associated proteins, including RelA, RelB and IKKα. After inhibiting the NF-κB pathway in these cells, the stemness phenotype was reduced, including the down-regulated expression levels of stemness genes and decreasing clonogenic capacity and tumorigenesis. 18 In the present study, we demonstrated that anlotinib suppressed NF-κB activity through down-regulating the phosphorylated levels of NF-κB and IκBα in PC-9-and HCC827-derived LCSCs. In order to further clarify whether regulating NF-κB activity was related to apoptosis induction and stemness phenotype attenuation in these cells, BAY11-7082, a specific NF-κB inhibitor, was employed. Previous studies suggested that BAY11-7082 suppressed the NF-κB activity in many types of cancers, including lung cancer, leukemic, prostate cancer and cervical cancer. [36][37][38][39] In this study, we demonstrated that BAY11-7082 induces apoptosis and attenuates stemness phenotype in PC-9-and HCC827-derived LCSCs.

| CON CLUS IONS
In summary, the current findings indicated that anlotinib exerts anticancer efficiency on PC-9-and HCC827-derived LCSCs in vitro and in vivo via apoptosis induction and stemness phenotypic attenuation. This phenomenon is associated with the suppression of NF-κB activity. Therefore, anlotinib may constitute a new agent for treating LCSCs, which needs to be investigated further.

CO N FLI C T O F I NTE R E S T
These authors declare that there is no conflict of interests regarding the publication of this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.