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 in vitro, sphere cells were collected by gentle centrifugation at 200 × g for 10 min, dissociated into single-cell suspensions, and cultured to allow sphere regeneration.

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′) (16). On-TARGET-plus control siRNAs (Thermo Fisher Scientific, Inc.) were used as control sequences.

Western blot analysis was carried out as described by Liu et al (17). Primary antibodies raised against AMPKα, p-AMPK, FoxO3a, p-FoxO3a, uPAR, ALDH1, Bmi1, SOX2 and β-actin were used. Cells were lysed in lysis buffer for 20 min at 4°C. Protein amounts were determined with the Bio-Rad assay system (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts (50 µg) of total protein were fractionated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes (PVDF) (Millipore, Billerica, MA, USA). Signals were detected using an ECL Advance Western blot analysis system (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). Experiments were carried out in triplicate (ImageJ v.1.84; National Institutes of Health, Bethesda, MD, USA).

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); n=number of measurements. Multiple groups were compared by one-way analysis of variance (ANOVA); comparisons of group means were performed by the LSD method for normally distributed variables. Two-tailed t-test was also used as appropriate. P<0.05 was considered statistically significant.

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 (Fig. 1A). To determine the capability of H446, H209 and H69 cells for self-renewal initiation, these cells were submitted to several serial passages. As shown in Fig. 1B, sphere-formation rate was highest for second-generation spheres at the third sphere-formation process in the H446 cell line compared with other cell lines or spheres of another generation. Therefore, second-generation spheres of H446 cells were considered to be lung cancer stem-like cells (LCSLCs), and used for subsequent experiments.

We next compared the in vitro oncogenic capabilities of LCSLCs and H446 cells by sphere- and colony formation assays. The results showed that the size and population of tumor spheres from LCSLCs were larger than the values obtained for H446 cells (Fig. 1C). In addition, the colony formation ability was significantly enhanced in LCSLCs compared with H446 cells (Fig. 1D). These data suggested that LCSLCs had the capacity of self-renewal and oncogenic capabilities in vitro, and were highly enriched in second-generation spheres of H446 cells.

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 (Fig. 1E).

To explore the role of AMPK/FoxO3a signaling in the in vitro carcinogenesis of H446-derived LCSLCs, the phosphorylated protein levels of AMPK, ACC and FoxO3a were determined. As shown in Fig. 1F, the phosphorylation levels of AMPK and ACC were lower, while FoxO3a protein phosphorylation was increased, in LCSLCs compared with H446 cells. These results suggest that inactivation of AMPK and FoxO3a were associated with carcinogenesis maintenance in vitro and the stem-like properties of H446-derived LCSLCs.

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 (Fig. 1G). Furthermore, H&E staining revealed similar histological characteristics in tumor xenografts derived from LCSLCs and H446 cells (Fig. 1H). Furthermore, uPAR protein amounts were higher in transplanted tumors of LCSLCs than those of H446 cells, as indicated by immunohistochemistry (Fig. 1H). These results provide sufficient evidence that H446 second-generation spheres possess LCSLC features such as increased in vitro carcinogenesis and in vivo tumorigenic potential.

Similar to our previous findings (17), this study demonstrated that the indicated concentrations (1.0, 3.0 and 10.0 µmol/l) of casticin suppressed self-renewal capability by reducing the sphere-formation rate in H446-derived LCSLCs (Fig. 2A), in a dose-dependent manner. In addition, colony formation assay on soft agar showed that casticin significantly inhibited the colony forming ability of H446-derived LCSLCs (Fig. 2B), in a dose-dependent manner. To further confirm the inhibitory effects of casticin on CSC characteristics, we next assessed the expression levels of SCLC CSC bio-markers, including uPAR and CD133, in H446-derived LCSLCs treated with casticin at the indicated concentrations. The results showed that the protein levels of uPAR and CD133 were dose-dependently reduced by casticin (Fig. 2C). Taken together, these findings demonstrated that casticin could inhibit in vitro carcinogenesis and CSC characteristics in H446-derived LCSCs.

To test the hypothesis that casticin inhibits in vitro carcinogenesis and CSC characteristics in H446-derived LCSCs through activation of AMPK/FoxO3a signaling, the effects of casticin on the phosphorylated protein levels of AMPK, ACC and FoxO3a were evaluated by western blot analysis. As shown in Fig. 2D, treatment with casticin resulted in significantly elevated phosphorylation levels of AMPK and ACC, and reduced FoxO3a phosphorylation, in a concentration-dependent manner. Collectively, these findings indicated that casticin inhibited in vitro carcinogenesis and CSC characteristics of H446-derived LCSCs, likely involving the activation of AMPK/FoxO3a signaling.

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 (18). Previous studies have demonstrated that casticin induces apoptosis through reactive oxygen species-mediated mitochondrial signaling pathways in cervical (19) and lung (20) cancers. Therefore, we hypothesized that the inhibitory effects of casticin on H446-derived LCSLCs may also involve AMPK activation. To test this, we firstly knocked down AMPK with siRNA in H446-derived LCSLCs, and evaluated the phosphorylated protein levels of AMPK, ACC and FoxO3a. As shown in Fig. 3A, transfection with AMPK siRNA resulted in reduced AMPK protein expression, decreased ACC phosphorylation, and increased phosphorylation levels of FoxO3a, compared with the untreated or control siRNA-treated H446-derived LCSLCs. Moreover, AMPK knockdown enhanced sphere and colony formation abilities, compared with the untreated or control siRNA-treated H446-derived LCSLCs (Fig. 3B and C). Accordingly, the protein expression levels of uPAR and CD133 were increased in the H446-derived LCSLCs transfected with AMPK siRNA compared with the untreated or control siRNA-treated counterparts (Fig. 3D). These results suggested that AMPK knockdown blocked AMPK/FoxO3a signaling, and enhanced in vitro carcinogenesis and CSC characteristics in H446-derived LCSLCs.

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 (Fig. 4A). In addition, AMPK knockdown also elevated the phosphorylation levels of FoxO3a, while attenuating the reduction of FoxO3a phosphorylation after treatment with casticin (3.0 µmol/l) in H446-derived LCSLCs (Fig. 4A). Furthermore, AMPK knockdown not only enhanced self-renewal and colony formation capabilities, but also counteracted the inhibitory effects of casticin on carcinogenesis in H446-derived LCSLCs in vitro (Fig. 4B and C). Meanwhile, transfection with AMPK siRNA resulted in increased uPAR and CD133 protein levels, reversing the inhibitory effects of casticin on uPAR and CD133 expression in H446-derived LCSLCs (Fig. 4D). These results suggested that AMPK activation may be upstream of FoxO3a activation in response to casticin stimulation 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 (21). To assess whether the in vitro effects of casticin on carcinogenesis and CSC characteristics in H446-derived LCSLCs involve AMPK/FoxO3a signaling, FoxO3a was silenced. As shown in Fig. 5A, FoxO3a knockdown mainly resulted in decreased FoxO3a protein levels, with no effects on AMPK and ACC levels or phosphorylation. Furthermore, FoxO3a knockdown increased sphere and colony formation rates (Fig. 5B and C) as well as uPAR and CD133 protein levels (Fig. 5D) in H446-derived LCSLCs. These results indicated that activation of FoxO3a could inhibit in vitro carcinogenesis and CSC characteristics in H446-derived LCSLCs, and FoxO3a may be a downstream target of AMPK.

To further assess whether the effects of casticin on in vitro carcinogenesis and CSC characteristics are mediated by AMPK/FoxO3a signaling, casticin (0 or 3.0 µmol/l) was administered to H446-derived LCSLCs transfected with FoxO3a or control siRNA. As expected, FoxO3a knockdown synergistically reduced FoxO3a protein phosphorylation, with slight effects on elevated AMPK and ACC phosphorylation levels associated with casticin (3.0 µmol/l) in H446-derived LCSLCs (Fig. 6A). As shown in Fig. 6B and C, FoxO3a silencing neutralized the inhibitory effects of casticin (3.0 µmol/l) on sphere and colony formation abilities in H446-derived LCSLCs. Consistently, FoxO3a knockdown weakened the decreased expression levels of uPAR and CD133 observed in H446-derived LCSLCs in response to treatment with casticin (3.0 µmol/l) (Fig. 6D). These findings clearly indicated that the inhibitory effects of casticin on in vitro carcinogenesis and CSC properties were mediated by AMPK/FoxO3a signaling in H446-derived LCSLCs.