The synergistic anticancer effect of formosanin C and polyphyllin VII based on caspase‐mediated cleavage of Beclin1 inhibiting autophagy and promoting apoptosis

Abstract Objectives Drug combination has a promising and potential development prospect in the treatment of various cancers. The objective of this study is to investigate the synergistic mechanisms of polyphyllin VII (PVII) and formosanin C (FC) in lung cancer. Materials and methods The combination of FC and PVII influenced on the apoptosis, autophagy, and the relative signalling pathways were analysed in lung cancer cells. Results The combination of FC and PVII demonstrated a concentration‐ dependent growth inhibition in human lung cancer cells. The combination index (CI) obtained from four lung cancer cells was smaller than 1. This synergistic antitumour effect was based on the increase of their single proapoptotic effect but inhibiting FC‐induced autophagy in NCI‐H460 cells. FC and PVII activated proapoptotic elements like cleaved‐caspase‐3, ‐8, and ‐9 to induce Beclin1 cleaved into Beclin1‐C which suppressed FC‐triggered autophagy and enhanced apoptosis. Conclusions Formosanin C and PVII showed a synergistic antitumour effect on lung cancer cells. The findings would provide the foundation for the use of combination drugs in the future.

Modern experiments discovered that RPS possessed two main structures of saponins including diosgenin and pennogenin. 8 Polyphyllin I 9 and formosanin C (FC) 10 belonging to the diosgenins, exhibited the anticancer activity mainly through inducing cellular apoptosis and activating autophagy. FC inhibited Bcl-2 and activated proapoptotic elements including Bax, caspase-2, 11 3, and -9. 12 Meanwhile, FC provided a potential inhibitive effect on pulmonary metastasis via repression of matrix metalloproteinases. 13 Polyphyllin VII (PVII) as a pennogenin, showed anticancer activity mainly through triggering G2/M cell cycle arrest and apoptosis based on a caspase-3dependent manner 14 and inducing mitochondria dysfunction. 15 With the wide application of complex mixtures in clinics, synergistic interactions play important roles in phytomedicine. In our previous study, RPS as a mixture, displayed a better antitumour effect than its monomers did. Two diosgenins, polyphyllin I and FC from RPS displayed a synergistic antitumour effect on hepatocarcinoma cells through increasing their single G1 phase arrest and mitochondria-dependent apoptotic pathway. 16 Recently, after further screening different kinds of saponins from RPS in the synergistic antitumour effect on lung cancer, FC and PVII exhibited different antitumour mechanisms and had a stronger synergistic antitumour activity in lung cancer cells. Therefore, it is of inherent importance to carry out their synergistic antitumour research.

| Reagents
Formosanin C and PVII were purchased from National Institute for the Control of Pharmaceutical and Biological Products, China.
Their batches were 111591-201604 and 111593-201604, respectively. The other reagents were commercially available and of analytical purity.

| MTT assay
Cell viability was determined by a colorimetric assay using 3-(4,5dimethyl-thiagol-2yl)-2,5-diplenyltertrazollium (MTT). Four kinds of cancer cells were seeded at a density of 1 × 10 4 /well in a complete growth medium in 96-well plates. The cells were incubated with the test compounds for 24 hours before the MTT assay. Then, a fresh solution of MTT (0.5 mg/mL) was added to each single well with a further incubation for 4 hours. Finally, the cells were dissolved with 100 μL of DMSO and then analysed in a multiwall plate reader at 570 nm (BioTek Instruments, Inc., Winooski, VT, USA).

| Colony formation assay
The NCI-H460 cells were digested with 0.25% trypsin and split into individual cells. Subsequently, 1000 cells were seeded into 3-mL culture dishes and maintained under standard culture conditions for 1 week.
When the colonies were visible to the naked eye, the cells were incubated with the test compounds for 5 days. Then, the colonies were stained with MTT (0.5 mg/mL) for 4 hours and observed under a microscope.

| Median effect analysis
For combination studies, MTT assays of FC and PVII at a fixed concentration ratio (1:1) were carried out for 24 hours treatment. The concentration-response curves of each agent for each cell line were obtained.
The interactions between drugs were evaluated by calculating Chou-Talalay combination indices (CI) using Compusyn software.

| Annexin-V/PI double-staining
An FITC annexin-V Apoptosis Detection Kit I (KeyGEN BioTECH, Nanjing, China) was used to detect apoptosis in human lung cancer cells after exposure to the test compounds. NCI-H460 cells were washed in phosphate-buffered saline thrice and resuspended in 100 μL of binding buffer with FITC annexin-V and PI (1 μL each). Quantification of apoptotic cells was determined using Annexin V-FITC and PI was used to distinguish necrotic cells.
The cells were washed in phosphate-buffered saline thrice and then fixed in 100% ice-cold methyl alcohol for 10 minutes. After treatment, the cells were washed in PBS thrice and stained with Hoechst 33258 (1 μg/mL) for 30 minutes. Cells were photographed by fluorescence microscope.

| Fluorescence imaging of cells
For AO staining, NCI-H460 cells (1 × 10 5 /well) were seeded and cultured at 37°C for 24 hours. After treatment with the test compounds of 24 hours, 2 μmol/L AO was added to the cells and incubated at 37°C for 15 minutes in the dark, the cells were washed three times with Dulbecco's phosphate-buffered saline. Images were captured and analysed using a fluorescence microscopy (Olympus, Tokyo, Japan).

| Immunofluorescence staining
Cells grown on glass coverslips were treated with the test compounds for 24 hours. Next, slides were fixed with 4% paraformaldehyde for 20 minutes and treated with 0.05% Triton X-100 for 15 minutes at RT. Thereafter, the coverslips were stained with LC3 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) (diluted in 5% BSA) for 3 hours at RT, then washed with PBS thrice, followed by incubation  with secondary antibodies at RT for 1 hour. Next, nuclei were stained with DAPI for 1 minute. Images were acquired using fluorescence microscopy.

| siRNA transfection
NCI-H460 cells were plated into six-well plates with 20%-30% confluency for 24 hours. Then, the cells were transfected with 100 nmol/L siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The siRNA targeting Beclin1 and the scramble control (NC) siRNA were designed, modified, and synthesized by Invitrogen.  were incubated with secondary anti-rabbit antibody at 1:5000 dilutions for 1 hour at RT. After washing, protein bands were visualized by Odyssey infrared imaging system (LI-COR Biotechnology, Lincoln, NE, USA). Western blot analyses were performed using standard procedures. All the experiments were repeated thrice.

| Statistical analysis
Statistical evaluation was conducted by using SPSS 17.0 for Windows package software. Data have been expressed as the means ± standard error mean (SEM). One-way variance analysis and Duncan multiple range test were used to determine significantly different groups.
P values less than 0.05 were considered as significant differences for all statistical calculations.

| FC and PVII exerted synergistic antiproliferative effects on lung cancer cells
FC and PVII ( Figure 1A) significantly inhibited cell viability in four lung cancer cell lines in a concentration-dependent manner. In order to share an equipotent effect of each partner, the combination ratios were designed to approximate the IC50 ratios. Colony formation assay indicated that there were the least cells in the combination group among four groups ( Figure 1B). Subsequently, different cells were exposed to various concentrations (0.25, 0.5, 1.0, 2.0, and 2.5 μmol/L) of FC, PVII or their combination (1:1 concentration) ( Table 1). The combination of FC and PVII showed a higher inhibitive rate than single-agent treatment in each cell (Table 2). Therefore, the combination induced synergistic effects in lung cancer cells, following evaluation of the CI through isobiologram analyses. The results showed that the CI values in four kinds of lung cancer cell lines were less than 1, indicating synergistic effects between two compounds.

| Combination of FC and PVII enhanced their single proapoptosis in NCI-H460 cells
To investigate whether the growth inhibition of combination was caused by cell apoptosis, Annexin V-FITC/PI double-staining and Hoechst 33258 staining were employed in H460 cells (Figure 2A

| Combination of FC and PVII inhibited autophagy induced by FC in NCI-H460 cells
According to previous studies, FC induced autophagy in H460 cells. 10 To understand the mechanism of combination, cellular morphology was observed using microscope. After 24-hours treatment with FC, numerous cytoplasmic vacuoles were formed. However, the number was decreased in the combination group ( Figure 3A). Acridine orange (AO) staining was used to identify autophagic acidic vesicles (AVOs) formation. Flow cytometer quantified the red fluorescence intensity of AVOs stained with AO. As a result, the combination group decreased the number and the fluorescence intensity of the autophagic acidic vesicles induced by FC ( Figure 3B). In addition, the formation of LC3 puncta was analysed by immunofluorescence staining. As shown in Figure 3C

| Caspases inhibitor potentiated the effects of FC or FC&PVII on the induction of autophagy and inhibition of apoptosis in NCI-H460 cells
To investigate the proapoptotic role of PVII in the synergistic effect of FC&PVII in NCI-H460 cells, a pan-caspase inhibitor Z-VAD-fmk was further applied. The results manifested that Z-VAD-fmk significantly decreased the protein levels of cleaved-caspase-3 and -9. However, it promoted the formation of autophagic vacuoles ( Figure 5A) and increased the autophagy protein levels of LC3II/LC3I and Beclin1 ( Figure 5B,C).
These results manifested that the balance between apoptosis and autophagy was associated with caspase regulation. Furthermore, combination of FC and Z-VAD-fmk-treated cells displayed the similar phenomenon to that in the combination of FC&PVII and Z-VAD-fmk groups. In this study, combination of FC and PVII demonstrated a concentration-dependent growth inhibition in human lung cancer cells.

| Combination of PVII & FC induced caspases
The CI values obtained from four lung cancer cells were smaller than 1, which confirmed a synergistic interaction in the combination of FC and PVII (Figure 1, Tables 1 and 2). In order to explain the synergistic antitumour mechanism of FC & PVII, cell morphology was observed under microscopes and fluorescence microscopes. As a result, combination of FC and PVII enhanced their single proapoptotic effects, but inhibited autophagy induced by FC in NCI-H460 cells.
To understand the proapoptosis and antiautophagy roles of PVII playing in the combination of FC and PVII, a pan-caspase inhibitor Z-VAD-fmk, the autophagy initiation protein class III PI3K inhibitor 3-MA and the later period lysosomal deacidification inhibitor CQ were used. 19 As a result, the autophagic inhibitors could not enhance the apoptosis induced by FC ( Figure 4). As we know, Bcl-2 as a central Atg5-Atg-12 with Fas-associated death domain protein can recruit and activate caspase-8 to induce extrinsic apoptotic signaling. 22 Autophagic inhibitors 3-MA and CQ significantly decreased apoptosis protein expression of cleaved-caspase-8 and slightly increased the ratio of Bcl-2/Bax. Therefore, inhibiting autophagy may suppress apoptosis induced by FC in NCI-H460 cells. However, the role of PVII played in the inhibition of autophagy was not the same as that of CQ or 3-MA.
Caspase inhibitor potentiated the effects of FC or FC&PVII on the induction of autophagy and inhibition of apoptosis in NCI-H460 cells ( Figure 5). As previously reported, PVII could trigger apoptosis via a caspase-3-dependent manner 14 and induce mitochondria dysfunction. 15 In this study, PVII also increased cleavage of caspase-3, -8, and -9. Previous studies showed that apoptosis induced cleavage of core proteins involved in autophagy (eg, Beclin1), which inhibited autophagy. The protein fragments further triggered apoptotic cell death. 23 Especially for caspases, they mediated cleavage of Beclin 1 to form Beclin1-C, which terminated Beclin 1 inducing autophagy and activated apoptosis. 17 As shown in Figure 6, combination of FC and PVII enhanced C-ter-

CO N FLI C T O F I NTE R E S T
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.