Leaves of A. muricata were purchased from Todam (Cheonan, Korea). ALPS were prepared following a previously described method (22). Briefly, 50 g of A. muricata leaf powder were extracted with 500 ml of deionized water at 100°C for 2 h. The extract was filtered through Whatman no. 4 filter paper then incubated with five volumes of 70% ethanol overnight at 4°C. The ethanol phase was centrifuged at 1,700 × g for 20 min to collect the precipitated polysaccharides, which were lyophilized (Hanil, Gwangju, Korea) and dissolved in sterile deionized water.

CP and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The fluorescein isothiocyanate (FITC)-conjugated annexin V/propidium iodide (PI) kit was purchased from BD Biosciences (San Diego, CA, USA). Monoclonal primary Abs against cleaved caspase-3 (#9661), cleaved caspase-8 (#9496), cleaved caspase-9 (#9501), cleaved poly (ADP-ribose) polymerase (PARP, #9541), B-cell lymphoma 2 (Bcl-2, #2870), Bcl-2-associated X protein (BAX, #2772), cytochrome c (#12959), and β-actin (#4970), and horseradish peroxidase-conjugated goat anti-rabbit (#7074) and anti-mouse secondary Abs (#91196) were obtained from Cell Signaling Technology (Danvers, MA, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFDA) and 3,3′-dihexyloxacarbocyanine (DiOC₆) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Human lung cancer cell lines (A549 and H460) and a murine macrophage cell line (RAW 264.7) were obtained from the Korea Cell Line Bank (Seoul, Korea). Cells were cultured in complete Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin (Gibco BRL), and maintained in a humidified chamber at 37°C with 5% CO₂.

A549, H460, and RAW 264.7 cells were seeded in complete DMEM into 96-well plates and allowed to grow to approximately 70–80% confluency at 37°C with 5% CO₂. The cells were treated with 0–1,000 µg/ml ALPS for 2 h and then incubated with CP (0, 10, 15, and 20 µM) for 24 h. Subsequently, the medium was replaced with MTT solution (0.5 mg/ml in complete DMEM) and incubation continued for 2 h. The solution was aspirated, and the formed formazan crystals were dissolved in 150 µl of dimethyl sulfoxide (Sigma-Aldrich) per well. Absorbance was measured at 570 nm using an Epoch microplate reader (BioTek Instruments, Winooski, VT, USA).

The extent of apoptosis was determined by flow cytometry using a FITC-conjugated annexin V/PI kit and a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. RAW 264.7 cells were incubated in a 48-well plate for 12 h and then treated with CP in the presence or absence of ALPS for 24 h. The cells were harvested and washed with phosphate-buffered saline (PBS; Gibco BRL), resuspended in the binding buffer, and stained with annexin V-FITC and PI for 15 min. The stained cells were analyzed using a FACSVerse™ flow cytometer (BD Biosciences). The TUNEL assay was performed using the DeadEnd™ fluorometric TUNEL system (Promega, Madison, WI, USA) following the manufacturer's instructions. Briefly, the cells were fixed with a 4% formaldehyde solution in PBS for 25 min, then permeabilized with 0.2% Triton X-100 in PBS for 5 min. After washing with PBS, the samples were incubated in the reaction buffer from the staining kit for 60 min. TUNEL-positive cells were analyzed using a FACSVerse™ flow cytometer and FlowJo software (version 10, BD Biosciences).

RAW 264.7 cells were cultured on glass slides for 12 h, then treated with CP in the presence or absence of ALPS for 24 h. The cells were fixed in 4% paraformaldehyde in PBS for 30 min, then permeabilized in 0.2% Triton X-100/PBS (Sigma-Aldrich, Darmstadt, Germany) for 5 min. After washing the slides twice using PBS and adding 100 µl of the equilibration buffer for 10 min at 4°C, the samples were incubated in 50 µl of the TdT reaction mixture for 1 h at 37°C in a humidified chamber in the dark. To stop the reaction, the glass ± diamidino-2-phenylindole was added in the mounting medium and TUNEL-positive cells were analyzed using a LSM510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany).

Five, seven-week-old (18±2 g) female C57BL/6 mice were purchased from Orient Bio (Seoul, Korea). They were acclimated to the temperature (25±2°C) and humidity (55±5%) of the housing unit and fed a sterile commercial mouse diet and water ad libitum. BMDMs were isolated from these mice following an established protocol (23). Specifically, after sacrifice by cervical dislocation, bone marrow cells were isolated from the femur and tibia. Erythrocytes were lysed using a red blood cell lysing buffer (Sigma-Aldrich). Thereafter, BMDMs were plated in a petri dish and differentiated for six days in complete DMEM containing 10 ng/ml macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN, USA). On day 3 of differentiation, 10 ml of the macrophage complete medium were added. After 6 days of differentiation, the cells were harvested and the F4/80⁺ and CD11b⁺ BMDM populations (>90% purity) were isolated with a FACSverse using anti-F4/80 and anti-CD11b Abs (BD Bioscience). The animal experiment was approved by the Institutional Animal Care and Use Committee of the Korea Atomic Energy Research Institute (KAERI–IACUC-2020-005).

On day 7 of differentiation, adherent BMDMs were harvested using a 0.25% trypsin-EDTA solution (Gibco BRL) and plated into 48-well plates in complete DMEM at a density of 10,000 cells/well. The cells were treated with CP in the presence or absence of ALPS for 24 h, then stained with annexin V/PI as described above.

RAW 264.7 cells or BMDMs were seeded into 6-well plates and treated with CP in the presence or absence of ALPS. The cells were lysed in RIPA buffer (Pierce) containing a protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). After centrifugation at 16,000 × g for 20 min at 4°C, the total protein concentration was determined in the supernatant using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The cell lysates (20 µg of protein) were resolved by 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the separated proteins were electrotransferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk and incubated with primary Abs (diluted 1:1,000) against cleaved caspases-3, −8, and −9, cleaved PARP, BAX, Bcl-2, cytochrome c, and β-actin overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary Abs (diluted 1:5,000) for 1 h. Protein bands were visualized using the Pierce ECL western blotting substrate (Thermo Fisher Scientific).

Intracellular ROS levels were measured using the H2DCFDA assay (24). After incubation with CP and ALPS, 10 µM H2DCFDA was added to the cells for 30 min at 37°C in the dark, and the cells were detached from the plates. After the cells were washed twice with PBS, the fluorescence intensity of the oxidized DCF was detected using a FACSVerse™ flow cytometer and FlowJo software.

Loss of the MTP was analyzed using DiOC₆. Cells were incubated with 10 nM DiOC₆ in fresh medium for 30 min at 37°C in the dark, washed, and resuspended in PBS. The fluorescence intensity of DiOC6 was detected using a FACSVerse™ flow cytometer and FlowJo software.

All experiments were repeated three times using triplicate wells. Statistical significance was analyzed by a one and two-way analysis of variance followed by Tukey's test using Prism version 8.0 software (GraphPad Software, San Diego, CA, USA). The results are expressed as means ± standard deviation. Values of *P<0.05, **P<0.01 and ***P<0.001 were considered statistically significant.

To identify the mitigating effect of ALPS in CP-induced cytotoxicity, RAW 264.7 macrophages- and human lung cancer cell lines (A549 and H460) were used to demonstrate the effect of ALPS on normal and cancer cells. To determine the appropriate concentration of ALPS, ALPS cytotoxicity was first evaluated against RAW 264.7 macrophages. As shown in Fig. 1A, ALPS did not exert cytotoxicity at the concentration range of 15.6–500 µg/ml in RAW 264.7 macrophages. Next, we investigated whether ALPS affected the viability of CP-treated lung cancer (A549 and H460) cells and RAW 264.7 macrophages. In A549 and H460 cells, both CP alone (10, 15, and 20 µM) and in combination with ALPS (15.6, 31.25, and 62.5 µg/ml) resulted in a concentration-dependent inhibition of tumor cell growth compared with that in the control (CP-untreated) group. These results were in agreement with supplement effect of ALPS on CP-induced ROS production and MTP loss in lung cancer cells (Figs. S1 and S2). Meanwhile, the CP-induced cytotoxicity was effectively suppressed by treatment of RAW 264.7 macrophages with ALPS at various concentrations (15.6, 31.25, and 62.5 µg/ml) compared with that in the CP alone group (Fig. 1B). Furthermore, at concentrations of ALPS higher than 62.5 µg/ml, the cytoprotective effect was similar to the concentration at 62.5 µg/ml of ALPS in cisplatin-treated RAW 264.7 macrophage (data not shown). Based on the results obtained, the appropriate concentrations for ALPS (31.25 and 62.5 µg/ml) and CP (15 µM) were determined and used in all subsequent experiments.

To further confirm that treatment with ALPS resulted in a concentration-dependent increase in the viability of CP-treated RAW 264.7 macrophage cells, the cytoprotective effects of ALPS against CP-induced apoptosis were evaluated using annexin V/PI or TUNEL staining. As shown in Fig. 2A, pretreatment with ALPS (31.25 and 62.5 µg/ml) resulted in a significant increase in cell viability compared with that in the CP only group (P<0.001). Furthermore, the CP only treated group showed an increased number of TUNEL-positive cells compared with that in the CP-untreated group, whereas the number was significantly (P<0.001) reduced by pretreatment with ALPS (31.25 and 62.5 µg/ml) in CP-treated RAW 264.7 macrophages (Fig. 2B). These results were consistent with the confocal microscopic analysis via TUNEL staining (Fig. 2C). Collectively, these results demonstrated that ALPS attenuated the apoptotic cell death of CP-treated RAW 264.7 macrophages.

To elucidate the possible molecular pathways involved in the cytoprotective action of ALPS against CP-induced apoptosis, we examined the expression of the proapoptotic BAX and antiapoptotic Bcl-2 proteins, as well as that of PARP, cytochrome c, caspases-3, −8, and −9, in the CP-treated RAW 264.7 macrophages. As shown in Fig. 3A and B, exposure to CP resulted in increased levels of cleaved caspases-3, −8, and −9 in RAW 264.7 macrophages. In contrast, the levels of cleaved caspases-3, −8, and −9 were noticeably reduced in the CP-treated cells after pretreatment with ALPS (31.25 and 62.5 µg/ml) (P<0.001). Furthermore, PARP cleavage was higher in the CP-treated group than in the control group, whereas pretreatment with ALPS significantly (P<0.001) suppressed PARP cleavage in CP-treated RAW 264.7 macrophages.

Next, we investigated the involvement of BAX, Bcl-2, and cytosolic cytochrome c in the cytotoxic effects observed in CP-treated RAW 264.7 macrophages. The CP only treated group showed upregulation of BAX and cytosolic cytochrome c and downregulation of Bcl-2, whereas pretreatment with ALPS (31.25 and 62.5 µg/ml) significantly (P<0.001) reduced the expression of BAX and cytosolic cytochrome c and increased Bcl-2 expression (Fig. 3C and D). These findings suggested that ALPS inhibited the apoptotic cascade involved in CP-induced cell death, thereby promoting macrophage survival through a cytoprotective action.

Next, we examined whether the cytoprotective action of ALPS (31.25 and 62.5 µg/ml) is associated with ROS generation and MTP loss in CP-treated macrophages. As shown in Fig. 4A, treatment of macrophages with CP (15 µM) resulted in an increase in the ROS levels compared with those in the control group, whereas this increase was significantly attenuated by pretreatment of macrophages with ALPS (31.25 and 62.5 µg/ml) (P<0.001). In addition, a significant MTP loss was observed in CP-treated RAW 264.7 macrophages (Fig. 4B), which was attenuated by pretreatment with ALPS (31.25 and 62.5 µg/ml) (P<0.001). These findings suggested that the ALPS-induced cytoprotective effect was due to the inhibition of the apoptotic cascade by reducing CP-induced ROS production and MTP loss.

The cytoprotective effects of ALPS against CP-induced apoptosis were further elucidated using normal primary BMDMs. Consistent with the results obtained using RAW 264.7 macrophages, pretreatment with ALPS (31.25 and 62.5 µg/ml) induced a significant increase in BMDM viability compared to that of BMDMs treated with CP alone (P<0.001; Fig. 5A). Additionally, pretreatment with ALPS (31.25 and 62.5 µg/ml) significantly inhibited the CP-triggered activation of caspases-3 (P<0.001), −8 (P<0.01), and −9 (P<0.05) in BMDMs, thereby attenuating the apoptotic cell death (Fig. 5B). These findings suggest that ALPS might act as an effective adjuvant therapy against the toxic side effects induced by the chemotherapeutic agents.