Contributed equally
Casticin, a natural polymethoxyflavone isolated from
Small cell lung cancer (SCLC), a poorly differentiated and highly aggressive tumor, constitutes approximately 15% of all lung cancers (
Emerging evidence suggests that cancer stem cells (CSCs), a subpopulation of tumor cells, have the properties of self-renewal, heterogeneous progeny, drug-resistance, and carcinogenesis
FOXO3a is considered an evolutionarily conserved transcription factor involved in various cellular processes, including cell cycle arrest, DNA repair and tumor suppression (
Yung
The present study showed that casticin inhibited
The human small cell lung cancer NCI-H446, H209 and H69 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Shanghai, China), 100 U/ml penicillin and 100 U/ml streptomycin, in a humidified atmosphere with 5% CO2 at 37°C. Casticin (purity ≥98%) was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China), as yellow crystals (molecular weight, 374.3 Da). It was dissolved in dimethyl-sulfoxide (DMSO) to prepare a 10 mmol/l stock solution, diluted in cell culture medium immediately before use. The following reagents were purchased from Hunan Clonetimes Biotech Co., Ltd. (Changsha, China): Antibodies against AMPKα (cat. no. 2532), p-AMPK (cat. no. 8324), ACC (cat. no. 9957), FoxO3a (cat. no. 2497), p-FoxO3a (cat. no. 9465), uPAR (cat. no. 9692) and CD133 (cat. no. 3570S) (Cell Signaling Technology, Inc., Danvers, MA. USA); antibodies targeting p-ACC (cat. no. sc-271965; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and human β-actin (cat. no. A4700; Sigma Chemicals; Merck KGaA, Darmstadt, Germany) were employed as well. Other reagents included Invitrogen™ Lipofectamine 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the growth supplements B-27 and N-2 (Invitrogen; Thermo Fisher Scientific, Inc.).
To obtain spheres, the cells were cultured in stem cell-conditioned medium (DMEM/F12 medium supplemented with 0.02× B27, 20 ng/ml EGF, 20 ng/ml bFGF, 0.4% BSA, 4 µg/ml insulin, 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) in ultra-low attachment 6-well plates (Corning Inc., Corning, NY, USA). When spheres reached ≥20 cells, the suspension cultures were passaged every six days. Spheres were counted in 10 different high power fields using an inverted microscope (Nikon TS100; Nikon, Tokyo, Japan).
For future generation of spheres
To determine the sphere-formation rate, the dissociated cells or second-generation spheres treated with casticin (final concentrations of 1.0, 3.0 and 10.0 µmol/l, respectively) were seeded at a density of 1,000 cells/ml in 6-well plates to generate new spheres. The total number of spheres was recorded after 6 days of culture. Sphere formation rate was calculated by dividing the total number of spheres formed by that of live cells seeded multiplied by 100.
Each well of a 6-well culture plate was coated with 2 ml bottom agar-medium mixture (DMEM, 10% FBS, and 0.6% agar). After solidification, 2 ml top agar-medium mixture (DMEM, 10% FBS, and 0.3% Noble agar; BD Difco™; BD Biosciences, Franklin Lakes, NJ, USA) containing 1,000 cells treated as described above were added. After 14 days, the colonies formed (≥20 cells) were counted under an inverted fluorescent microscope (Olympus CK40; Olympus Corp., Tokyo, Japan), with the representative views imaged. Colony formation rate was calculated by dividing the total number of colonies formed by that of live cells seeded multiplied by 100.
Dissociated second-generation spheres were plated at 2.5×105 cells/well in 6-well plates. After 24 h, the siRNA-negative control (si-NC; Santa Cruz Biotechnology, Inc.) and AMPK- or FoxO3a-specific siRNAs (siAMPK or siFoxO3a; Shanghai GenePharma Co., Ltd., Shanghai, China) were transfected into cells, respectively, using Invitrogen™ Lipofectamine 2000 reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Separate siRNAs were used for FoxO3A (5′-GACAAUAGCAACAAGUAUA-3′) and AMPK (5′-GAGGAGCUAUUUGAUUA-3′) (
Western blot analysis was carried out as described by Liu
Four micron-thick tissue sections were immunostained with uPAR-specific antibody. Immunostaining was performed using a Ventana Discovery Ultra (Ventana Medical Systems, Tucson, AZ, USA). Antigen retrieval was performed using CC1 for 40 min at 95°C. IHC staining was followed by hematoxylin counterstaining. Slides were rinsed, dehydrated though alcohol and xylene and coverslipped.
Twenty-four pathogen-free BALB/c-nu female mice (13–15 g) aged 4 weeks were purchased from the Animal Institute of the Chinese Academy of Medical Science (CAMS). All animal studies were performed in accordance with the standard protocols, and approved by the Ethics Committee of Hunan Normal University and the Committee of Experimental Animal Feeding and Management. Mice were randomly divided into 3 groups (4 mice/group), and maintained under standard conditions. Varying amounts of H446 cells (103, 104 and 105, respectively) were subcutaneously injected into 4-week-old female nude mice in the left flank; in parallel, second generation sphere-derived cells (LCSLCs, 102, 103 and 104, respectively) were subcutaneously injected into the right flank. Tumor growth was monitored visually every week, and the maximum tumor volume allowed was consistent with the IACUC guidelines (diameter, 1.5 cm; area, 1.8 cm2; volume 1.8 cm3). Tumor volumes were calculated in accordance with the formula: V (transplanted tumor volume, mm3) = L (longest diameter, mm) × W (minimum diameter, mm)2 × 0.5. After 8 weeks of tumor growth, the mice were euthanized using cervical vertebra luxation. The obtained tumor tissues were fixed in formalin and embedded in paraffin. Hematoxylin and eosin (H&E) staining and immunohistochemical analysis were performed to assess tumor histology and tumor markers in the mouse xenografts.
SPSS 20.0 software (IBM Corp., Armonk, NY, USA) was used for analysis. Data are expressed as the mean ± standard deviation (SD);
To evaluate the sphere-forming capabilities of the SCLC H446, H209 and H69 cell lines, sphere-formation rates of the three cell lines were assessed. The results showed that the sphere-forming capability of H446 cells was higher than that of both H209 and H69 cell lines (
We next compared the
To further confirm the stem-like properties of LCSLCs, the protein expression levels of SCLC CSC-related markers were assessed in LCSLCs and H446 cells. Western blot analysis demonstrated that the protein expression levels of uPAR and CD133 were higher in the LCSLCs than these levels in the H446 cells (
To explore the role of AMPK/FoxO3a signaling in the
In addition, the abilities of LCSLCs and H446 cells to form tumors in BALB/c-nu mice were assessed. As many as 1×105 H446 cells were required to initiate stable tumor formation for 23–42 days after injection, while, as few as 1×103 LCSLCs were sufficient to generate visible tumors only 19–28 days post-injection (
Similar to our previous findings (
To test the hypothesis that casticin inhibits
AMPK is a central cellular energetic biosensor that regulates a broad array of cellular metabolic routes activated by nutrient deprivation, mitochondrial dysfunction, oxidative stress and cytokines (
To assess whether the inhibitory effects of casticin on oncogenicity in H446-derived LCSLCs is affected by AMPK regulation, casticin (0 or 3.0 µmol/l) was administered to H446-derived LCSLCs transfected with AMPK- and control siRNAs, respectively. Compared with the control siRNA group, AMPK silencing not only reduced AMPK levels and ACC phosphorylation, but also antagonized elevated phosphorylation levels of AMPK and ACC in response to casticin administration (3.0 µmol/l) in H446-derived LCSLCs (
FoxO3a, a downstream effector of AMPK, is associated with a variety of cell processes, including cell cycle progression, apoptosis, stress, detoxification, DNA repair, glucose metabolism and differentiation (
To further assess whether the effects of casticin on
The present study confirmed that casticin reduced the
He
Given that casticin suppresses cell growth in ovarian cancer (SKOV3) cells through FOXO3a activation (
It was reported that AMPK acts as an upstream regulator of Foxo3a (
Overexpression of uPAR is strongly correlated with the malignant cancer phenotype and poor prognosis (
In summary, this study firstly provided evidence that AMPK plays a role in regulating
Not applicable.
The present study was supported by the Project of NSFC (nos. 30760248 and 81172375), the Project of Scientific Research of Hunan Province, granted by the Health and Family Planning Commission of Hunan Province (no. B2013-098).
The datasets used during the present study are available from the corresponding author upon reasonable request.
Professor JC conceived and designed the study. QG, XC and XY performed the experiments. QG and XC wrote the manuscript. Professor JC and WZ reviewed and edited the manuscript. Professor WZ was also involved in the conception of the study. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.
All animal studies were performed in accordance with the standard protocols, and approved by the Ethics Committee of Hunan Normal University and the Committee of Experimental Animal Feeding and Management.
Not applicable.
The authors declare that they have no conflict of interest.
Effects of casticin on
Effects of AMPK siRNA on
Effects of casticin on
Effects of FoxO3a silencing on
Effects of casticin on