LBP (Hainan Changan International Pharmaceutical Co., Ltd.) was dissolved in dimethyl sulfoxide (DMSO) to obtain 2.5 mM stock solutions (in vitro) or in physiological saline (in vivo). The stock solutions were kept frozen in aliquots at −20°C. DMSO, N-acetyl-L-cysteine (NAC), and SB203580 were obtained from Sigma-Aldrich (St. Louis, MO, USA). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). The Annexin V-FITC Apoptosis Detection Kit was purchased from BD (Franklin Lakes, NJ, USA). The TUNEL staining kit was purchased from Roche (Basel, Switzerland). The ROS detection kit was purchased from Beyotime Institute of Biotechnology (Jiangsu, People's Republic of China). Antibodies against caspase-3, caspase-8, caspase-9, Bcl-2, p53, p-p38MAPK, poly-adenosine diphosphate (ADP) ribose polymerase (PARP), and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibody against Bax was purchased from Proteintech Group Inc. (Chicago, IL, USA). GIPZ Lentiviral shRNA was purchased from Horizon Discovery (Cambridge, UK).

A549, SK-MES-1, NCI-H1299, and 293T cells were acquired from Cobioer (Jiangsu, People's Republic of China). SK-MES-1 cells were cultured in MEM medium supplemented with 10% (v/v) heat-inactivated newborn calf serum (Thermo Fisher Scientific) with 1 ×10⁵ U/L penicillin G and 1 ×10⁵ U/L streptomycin in a humidified incubator at 37°C and 5% CO₂. NCI-H1299, A549, and A549/control-shRNA cells were cultured in RPMI l640 medium supplemented with 10% (v/v) heat-inactivated newborn calf serum (Thermo Fisher Scientific) with 1 ×10⁵ U/L penicillin G and 1 ×10⁵ U/L streptomycin in a humidified incubator at 37°C and 5% CO₂. 293T cells were cultured in DMEM medium supplemented with 10% (v/v) heat-inactivated newborn calf serum (Thermo Fisher Scientific) with 1 ×10⁵ U/L penicillin G and 1 ×10⁵ U/L streptomycin in a humidified incubator at 37°C and 5% CO₂.

The cytotoxic effect of LBP on NSCLC was determined by the CCK-8 assay. Cells were seeded in a 96-well culture plate at a density of 4 ×10⁴ cells/well and were cultured prior to exposure to a series of concentrations of LBP for 24, 48, and 72 h. A total of 10 μl of the CCK-8 solution was added to each well and incubated for an additional 4 h. The absorbance was measured at a 450-nm wavelength using the ELX 800 Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The measurement was performed three times for each concentration. The 50% inhibitory concentration (IC₅₀) was determined by non-linear regression fit of the mean values of the data obtained.

The effect of LBP on human A549 cell cycle distribution was determined by FCM analysis. Approximately 3 ×10⁵ cells were harvested at room temperature after pretreatment with various LBP concentrations for 24 h. Cells were then collected, washed twice with phosphate-buffered saline (PBS), and fixed with ice-cold 70% ethanol at 4°C overnight. After centrifugation, cells were resuspended in PBS containing 50 μg/ml RNase and incubated at 37°C for 30 min. Then, the cells were stained with 100 μl of 100 μg/ml propidium iodide (PI) and analyzed by a flow cytometer (C6; BD). The cell cycle distribution was analyzed according to the standard procedures. The distribution of cell cycle phases (G₀/G₁, S, or G₂/M phase) was determined using Flow Jo.

Approximately 3 ×10⁵ A549 cells/L were harvested after pretreatment with various reagents for 24, 48, and 72 h. Then, cells were trypsinized with EDTA-free trypsinogen, washed twice in PBS, and resuspended in 400 μl of 1× binding buffer. Next, 5 μl of annexin V-FITC and 5 μl of PI were added. After incubation at room temperature for 15 min in the dark, cells were then analyzed by FCM (C6; BD).

A549 cells were seeded in a 96-well culture plate at a density of 3 ×10⁵ cells/well, with a coverslip (containing poly-lysine) in each well. Wells were incubated overnight and assigned to groups treated with 0, 12, or 24 μM LBP. After 24 h of treatment, cells were washed with PBS and fixed with formaldehyde for 30 min. Then, cells were again washed with PBS three times for 5 min each time. To perform the staining, 2.5 μg/ml DAPI was added in the dark for 10 min. After staining, cells were again washed three times with PBS for 5 min each time. Then, 10 μl of antifade was applied on the slide before a cell-containing slip was placed on the slide. Samples were then observed with a fluorescence microscope (400×) and photographed.

Western blot was performed as previously described (25). The code of caspase3 is #9662 (CST). The code of caspase9 is #9502 (CST). The code of caspase8 is #9494 (CST). The code of p-p38MAPK is #4511 (CST). The code of PARP is #9532 (CST). The code of p53 is #2527 (CST). The code of Bcl-2 is #2870 (CST). The code of GAPDH is #5174 (CST). The code of Bax is #50599-2-Ig (Proteintech). All primary antibodies and secondary were diluted to 1000 ×.

Five-weeks-old male BALB/Ca nude mice were purchased from Shanghai Laboratory Animal Center (Shanghai, China). A xenograft of A549 cells was established by inoculating viable A549 cells (10⁷ cells/100 μl PBS per mouse) into the right flanks of the nude mice. When the average tumor volume reached ~100 mm³, the nude mice were randomly divided into two groups (5 mice per group). The experimental group was treated with LBP (d1, d8, 12 mg/kg) via the tail vein, and the control group was treated with saline (d1, d8, and saline only) via the tail vein. The body weight of each mouse was recorded twice a week. Tumor size was measured every other day. Tumor volume was calculated with a caliper (calculated volume = shortest diameter² × longest diameter/2). The mice were sacrificed after 21 days, and the tumors were excised and stored at −80°C until further analysis. The relative tumor volume (RTV) of each mouse was determined by the formula RTV = V_t/V₀, where V_t is the volume at each measurement and V₀ is the volume at the initial treatment. The therapeutic effect of a given compound was expressed in terms of T/C (%), which was calculated by the formula T/C (%) = mean RTV of the treated group/mean RTV of the control group ×100%. All of the animal experiments were conducted following protocols approved by the animal ethics committee of the Medical School, Southeast University, and animal care was provided in accordance with institutional guidelines.

Formalin-fixed tumor tissues were embedded in paraffin before being sectioned. A TUNEL system was used to evaluate apoptosis in the tumor sections that were placed on slides according to the manufacturer's protocol. Tissue sections were analyzed to detect the localized green fluorescence of the apoptotic cells and the blue fluorescence of the cell nuclei. Images were acquired and photographed using an Olympus IX51 fluorescence microscope (400×).

Intracellular hydrogen peroxide levels were monitored by FCM after staining with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Briefly, cells in a logarithmic growth phase (3 ×10⁵ cells in each well) were treated with various reagents for various periods of time and were then labeled with 2.5 μM DCFH-DA for 20 min. Next, the cells were trypsinized, washed with PBS, and then analyzed by FCM (Becton Dickinson). The percentage of cells displaying increased dye uptake was used to reflect an increase in ROS level.

Specific shRNA and control shRNA were designed and synthesized by Nanjing Kebai Biotech. Co. Ltd. (Nanjing, China). A BLAST search was performed with the National Center for Biotechnology Information (NCBI) database to ensure that the shRNA constructs were targeting only human p53. The sequence of the p53 shRNA was 5′-TACACATGTAGTTGTAGTG-3′, while the sequence of the nonsense-p53 shRNA (NS-p53-shRNA) was 5′-TTCTCCGAACGTGTCACGT-3′. The oligonucleotides were annealed and cloned into the pGIPZ-CMV-GFP-Puro, as described by the manufacturer. A total of 2.5 μg of pGIPZ constructs containing shRNAs and 7.5 μg of packaging plasmid were used to transfect 293T cells following the calcium phosphate precipitation method. After 48 h, lentiviruses containing targeted gene shRNA were collected and used to transfect A549 cells according to the manufacturer's instructions.

Statistical analysis was performed using the SPSS 22.0 software (IBM Corporation, Armonk, NY) package for Windows. Statistical significance was calculated using unpaired Student's t-test, with P < 0.05 indicating statistical significance.

The CCK-8 assay was performed to evaluate the effects of LBP on the proliferation of A549, NCI-H1299, and SK-MES-1 cells. The cells were treated with LBP at concentrations of 270, 90, 30, 10, 3.3, 1.1, 0.37, or 0.123 μM for 24, 48, or 72 h. As shown in Figure 1B, LBP reduced the proliferation of cells in a dose- and time-dependent manner. The IC₅₀ values of LBP for SK-MES-1 cells were 63.47 ± 1.03, 15.77 ± 1.09, and 12.0 ± 1.08 μM when the cells were treated for 24, 48, and 72 h, respectively. The IC₅₀ values for NCI-H1299 cells were 67.26 ± 1.06, 17.98 ± 1.05, and 7.85 ± 1.01 μM for the 24-, 48-, and 72-h treatment times, respectively, while the IC₅₀ values for A549 cells were 11.69 ± 1.05, 5.02 ± 1.11, and 2.43 ± 1.01 μM for the 24-, 48-, and 72-h treatment times, respectively. It seems that LBP had a notably low IC₅₀ in A549 cells with wild-type p53. Therefore, the A549 cell line was chosen for the next experiments to investigate the anticancer mechanism of LBP. Based on the IC₅₀ of LBP, 6 and 12 μM were selected to be the experimental concentrations for the cell cycle arrest experiments, while 12 and 24 μM concentrations were selected for apoptosis detection, DAPI stain, and Western blot of A549 cells.

FCM analysis was performed to assess the cell cycle of cells treated with low concentrations of LBP (6 and 12 μM). After 24 h of treatment, 72.03 ± 1.53% of the cells remained in G1 phase under 6 μM LBP treatment (Figure 1C-b), while 73.78 ± 1.26% of the cells remained in G1 phase under 12 μM LBP treatment (Figure 1C-c). In the control group (Figure 1C-a), only 47.47 ± 1.59% of cells were detected in this phase. Additionally, the percentage of cells in the G2 or S phases was strikingly decreased in the experimental groups (P < 0.05). Our data showed that LBP could induce G1 phase cell cycle arrest at both 6 and 12 μM concentrations.

The Annexin V-FITC/PI Apoptosis Detection Kit was used to quantitatively determine whether LBP could induce A-549 cell apoptosis. The result showed that after treatment with 12 μM LBP for 24 h, 20.2 ± 1.3% of the experimental A549 cells developed apoptosis, while 44.5 ± 1.6% of the cells were affected at a concentration of 24 μM LBP. After we doubled the treatment time, apoptotic proportion of A549 cells treated with LBP at concentrations of 12 and 24 μM increased to 55.3 ± 1.2% and 70.0 ± 0.9%, respectively, suggesting that LBP could induce A549 cell apoptosis in a time- and dose-dependent manner (P < 0.05, Figure 2A). Next, we performed DAPI staining to observe the morphological changes of the cell nuclei during the process of LBP-induced apoptosis. After being treated with 12 μM LBP for 24 h, nuclei were observed to be condensed and deformed. When cells were treated with 24 μM LBP for 24 h, fewer nuclei were found intact in the same field of vision than were found in the cells treated with 12 μM LBP, and apoptosis bodies were found (Figure 2B). We then performed Western blot to study the expression levels of apoptosis-related proteins. After being treated with LBP at 12 or 24 μM LBP for 24 h, expression levels of clv-PARP, Bax, and clv-caspase-3,8,9 were upregulated and Bcl-2 was downregulated all in a dose-dependent manner (P < 0.05, Figure 2C).

To study LBP's antitumor efficacy against A549 cells in vivo, we further generated a model of nude mice bearing human A549 tumors. The data showed that the experimental group weighed slightly less than the control group did, but there was no significant difference (P > 0.05). The mice lost some weight right after each time they were treated with LBP, which indicated that LBP might cause a mild gastrointestinal reaction (Figure 2D-a). By the end of the experiment, the average of the LBP group tumor was 451 ± 113 mm³ while it was 936 ± 205 mm³ in the control group (Figure 2D-b). The T/C value was 55.2%, and the tumor inhibition rate was 47.9% and Supplementary Figures 1, 2 (Figures 2D-c,d), suggesting that LBP could inhibit the growth of the A549 tumor. TUNEL/DAPI staining indicated the apoptosis rate of 15.5 ± 2.1% in the treatment group, whereas the percentage was only 4.8 ± 0.5% in the control group (P < 0.05) (Figures 2E-a,b). Taken together, our results indicated that LBP inhibited the growth of A549 tumors in mice by inducing the apoptosis.

To test whether ROS are involved in the pathway through which LBP induces A549 cell apoptosis, we examined ROS levels in A549 cells after treating the cells with 24 μM LBP. We performed the ROS analysis with both DCFH-DA staining analysis with a fluorescence microscope and FCM. From the resulting images, it can be inferred that ROS release increased as the treatment time increased (Figures 3A–G), with an apparent burst at 6 h (P < 0.05) (Figure 3H). FCM analysis also confirmed this finding (P < 0.05) (Figure 3I).

To determine whether LBP-induced apoptosis requires p53, stable cell lines transfected with the plasmid-based shRNA expression system targeting p53 mRNA were established, and the sensitivity of these cell lines to LBP was then surveyed. As shown in Figure 4A, the p53 protein level of A549/p53-shRNA was efficiently knocked down compared to the expression in the A549 parental control or A549/NS-shRNA (vector control) cells. Subsequently, A549/NS-shRNA and A549/p53-shRNA cells were exposed to LBP at concentrations ranging from 0.0123 to 90 μM for 24, 48, or 72 h (Figure 4B). The IC₅₀ of LBP for A549/p53-shRNA cells was 39.54 ± 1.02 μM for the 24-h treatment, 27.89 ± 1.13 μM for the 48-h treatment, and 10.93 ± 1.08 μM for the 72-h treatment. However, the IC₅₀ of LBP for A549/NS-shRNA cells was 11.48 ± 1.12 μM for the 24-h treatment, 5.14 ± 0.94 μM for the 48-h treatment, and 2.49 ± 0.90 μM for the 72-h treatment. Compared to the A549 cells, the A549/NS-shRNA cells showed similar sensitivity to LBP (P > 0.05). Hence, A549/p53-shRNA cells with p53 knockdown became less sensitive to LBP, as shown by their higher IC₅₀ values (P < 0.05). Sequentially, FCM analysis in association with annexin V-PI double staining was performed to investigate the apoptosis of A549/NS-shRNA and A549/p53-shRNA cells treated with LBP. The results showed that after 24 h, 38.6 ± 1.1% of the 24 μM-LBP-treated A549/NS-shRNA24h cells underwent apoptosis, but no significant apoptosis observed in A549/p53-shRNA cells (Figure 4C). Our findings suggested that A549/p53-shRNA cells were more resistant to LBP-induced apoptosis than A549/NS-shRNA cells (P < 0.05). To further evaluate whether LBP could also modify the expression level of p38MAPK, expression levels of p53 and p-p38MAPK were measured in A549/NS-shRNA and A549/p53-shRNA cells that were treated with 24 μM LBP for 24 h. As shown in Figure 4D, LBP could upregulate the expression levels of p53 and p-p38MAPK in A549/NS-shRNA cells (P < 0.05) but not in A549/p53-shRNA cells (P > 0.05). Interestingly, p-p38MAPK was found to have a higher expression level in A549/p53-shRNA cells than in A549/NS-shRNA cells with no LBP treatment.

Based on previous results, we applied an ROS inhibiting agent, NAC, to determine whether ROS are crucial for LBP-induced apoptosis. After pretreatment with NAC, cells were incubated with 24 μM LBP for 24 h, followed by FCM. As shown in Figure 5A, the apoptosis rate was reduced from 40.7 ± 2.4% to 9.6 ± 2.1% (P < 0.05). Our findings suggested that ROS participated in the process of LBP-induced apoptosis.

With DCFH-DA staining followed by FCM, we measured the ROS levels in A549/NS-shRNA and A549/p53-shRNA cells treated with LBP at a concentration of 24 μM for 6 h. While only a slight upregulation of ROS release occurred in the A549/p53-shRNA cells, A549/NS-shRNA cells generated a much higher level of ROS under LBP treatment. The percentage of ROS released was significantly higher in the A549/NS-shRNA cells than in the A549/p53-shRNA cells (P < 0.05, Figure 5B). NAC can partially suppress LBP-induced ROS release (P < 0.05, Figure 5C). To investigate the precise connection between p53, ROS, and p38MAPK in the pathway of LBP-induced apoptosis, we applied NAC and SB203580, a p38MAPK inhibiting agent, to the cells before LBP treatment to monitor the expression levels of p53 and p38MAPK. The results were shown in Figure 5D. It is implied that LBP at a concentration of 24 μM could upregulate the expression of p53 and p-p38MAPK (P < 0.05). Treatment with NAC prevented p-p38MAPK activation (P < 0.05), while p53 levels remained unchanged (P > 0.05). This result suggested that the p53/ROS/p38MAPK pathway was involved in the LBP-induced apoptosis.

P38MAPK plays vital roles in many stress reaction pathways in human cells. P38MAPK has been reported to be involved in DDP's antitumor mechanism (22). In our experiment, LBP at a concentration of 24 μM can induce a significant upregulation of p-p38MAPK in A549 cells (P < 0.05), which was inhibited by the application of SB203580 (P > 0.05, Figure 6D). To further examine the role of p38MAPK in LBP-induced A549 cell apoptosis, we applied SB203580 to the cells before they were treated with 24 μM LBP for 24 h followed by FCM for apoptosis analysis. The results showed that pretreatment with SB203580 reduced the LBP-induced apoptosis rate in A549 cells from 40.7 ± 2.4% to 18.1 ± 1.6% (P < 0.05, Figure 6A). Our findings indicated that p38MAPK participates in the process of LBP-induced apoptosis. Next, we performed DCFH-DA staining followed by FCM to detect ROS accumulation in A549 cells treated with or without SB203580 before LBP treatment. The results showed that SB203580 failed to alleviate the accumulation of ROS induced by LBP (P > 0.05, Figure 6B). Moreover, our findings indicated that SB203580 could not inhibit the upregulation of p53 triggered by 24 μM LBP (P > 0.05, Figure 6C).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.