Human BC cell lines J82 and UMUC3 (provided by Dr. P Guo) were cultured in Dulbecco modified Eagle medium supplemented (Hyclone, Logan, UT) with 10% of BI serum (FBS) (Hyclone, Logan, UT) and 1% of penicillin-streptomycin at 37°C, in humidified air containing 5% of CO₂.

4MOD (98% purity, MW = 254.28) was isolated and purified from Dalbergia sissoo Roxb. in our preliminary study (Ran et al., 2019). and it prepared as a stock solution of 50 mM in dimethyl sulfoxide (DMSO). SC79, and CQ (Selleck-Biotool, Shanghai, China) were prepared as a stock solution of 50 mM in PBS. They were diluted in dimethyl sulfoxide in a range of concentrations. Antibodies against Akt, ERK, LC3, GAPDH and β-actin were purchased from Cell Signaling Technology (Cell Signaling, Beverly, MA). An Apoptosis Detection kit (FITC Annexin V) was purchased from Beyotime (CS, Hunan, China). The TUNEL Assay Kit was obtained from Elabscience. MA.

Cell proliferation was determined by using a Cell Counting Kit-8 assay (CCK-8, Meilunbio, Dalian, China). Cells were seeded into a 96-well plate (6 × 10³ cells/well) with 100 μL of culture medium. After 24 h, cells were treated with different drugs and incubated for 72 h. 10 μL CCK-8 solution was added to each well and cells were incubated at 37°C for 2 h. Light absorbance was measured at 450 nm using a Dynatech MR5000 plate reader.

A total of 6×10³ cells were seeded into 24-well dishes in 0.5 ml of medium for 24 h. Cells were further treated with different drugs, and then maintained for 6–8 days in a CO₂ incubator. Finally, the cells were fixed with 10% formaldehyde solution and then stained with 0.1% crystal violet, each experiment was performed in triplicate. The absorbance was measured using a microplate reader at 550-nm wavelength.

Transwell assay was used to analyze the impacts of different concentrations of 4MOD (0 μM, 5.0 μM, 10 μM) on cell migration and invasion of BC cells by applying a Transwell plate (Corning, United States) according to the manufacturer’s guidance. Briefly, the upper chambers were plated with around 1×10⁵ cells. Then the cells were fixed in paraformaldehyde (4%) and dyed using crystal violet. Invaded and migrated cells were recorded and calculated.

A total of 5×10³ cells were seeded onto a 12-well plate and allowed to reach full confluence. The monolayer was wounded using a cocktail stick. Cells were incubated with serum-free MEM medium, for the time period as stated. Digital images were taken at times 0, 24 and 48 h. The mean area was calculated using ImageJ software. The experiments were repeated three times.

Apoptosis was analyzed using a TUNEL Apoptosis Detection kit (Elabscience, Institute of Biotechnology) according to the manufacturer’s protocol. The nuclei were counterstained with DAPI for 5 min at room temperature in the dark and the slides were then mounted with anti-fade mounting medium. The levels of apoptosis were estimated as the ratio of the number of TUNEL-positive cells to the total number of DAPI-positive cells using a fluorescence microscope (magnification, ×200; Olympus Corporation).

Apoptosis was detected by flow cytometry via the examination of altered plasma membrane phospholipid packing by lipophilic dye Annexin V. Briefly, treated cells were harvested by trypsin, washed twice with PBS, and then resuspended in binding buffer at a concentration of 5.0 × 10⁵ cells/mL according to the manufacturer’s instruction. Thereafter, 5 μL of Annexin V-FITC and 10 μL of propidium iodide were added into 100 μL of cell suspension and incubated for 30 min at room temperature in the dark. After adding 300 μL of binding buffer, labeled cells were counted by flow cytometry within 30 min. All early apoptotic cells (Annexin V-positive and propidium iodide-negative), necrotic/late apoptotic cells (double positive), as well as living cells (double negative) were detected by flow cytometry on a FACS Calibur flow cytometer.

Total RNA was isolated using a Total RNA Extraction kit (GeneCopoeia™, Guangzhou, China). Reverse transcription into cDNA was performed using a SureScript™ First-Stand cDNA Synthesis Kit according to the User Manual (GeneCopoeia™, Guangzhou, China) and the cDNA was amplified using a SYBR^® Green qPCR Mix 2.0 (GeneCopoeia™, Guangzhou, China). The 2^−ΔΔCt method was used to perform analysis with 18 s RNA as the reference. The primers of LC3 were purchased from GeneCopoeia™. Data analysis was performed with Applied Biosystems Quant Studio 3 Real-time PCR System (Thermo Fisher Scientific, United States).

Western blot assessment was performed using regular procedures. Primary antibody was added in bovine serum albumin (BSA) and allowed to incubate overnight at 4°C, washed with TBST for six times (10 min per time). The secondary antibodies (CST, United States) were used for hatching the membranes 1 h at room temperature. The Pierce Super Signal chemiluminescent substrate (Rockford, IL, United States) was added followed by the visualization with a Fluorescentand Chemiluminescence Gel Imaging System (Jiapeng, ShangHai, China). Band intensity data were quantified using ImageJ.

Data were expressed as mean ± SD, and the statistical analysis was conducted using GraphPad prism 9. The unpaired Student’s t-test was used to compare two groups, and the one-way ANOVA was used to compare among multiple groups. *p < 0.05, **p < 0.01 was considered as statistically significant.

We used Cell Counting Kit-8 analysis to verify whether 4MOD affects the proliferation of BC cells in vitro. We exposed J82 and UMCU3 cells to various concentrations of 4MOD (0 μM, 2.5 μM, 5.0 μM, 10 μM, 20 μM). It was found that 4MOD inhibited cell proliferation in a dose-dependent manner (Figure 1A; Table 1). The IC₅₀ values of 4MOD induced inhibition at various concentrations were 8.17 μM, 14.5 μM on bladder cancer cells, respectively (Figure 1A; Table 1). Similarly, the colony formation was decreased by treatment with 0–20 μM 4MOD (Figures 1B,C). The J82 is the more sensitive cell line which is consistent with the results of CCK8 assay.

Cell scratch assay and Transwell assay were used to analyze the effect of 4MOD on the migration and invasion of BC cells. As shown in Figure 2, 4MOD-treated cells were remarkably enhanced the wound healing proportion than that of control in J82 and UMUC3 cells at 24 and 48 h (Figures 2A–D), Similarly, Transwell assays results showed that the invasion was decreased in the cells treated with 0–20 μM 4MOD (Figures 2E,F), suggesting that 4MOD can decrease the migration and invasion of BC cells in vitro.

As shown in Figure 3, compared to the control group, the cellular apoptosis was remarkably increased in the cells treated with 20.0 µM 4MOD, as demonstrated by both FITC and PI staining (Figures 3A,B). Consistently, as we can see in Figure 3C under a fluorescence microscope, 4MOD-treated cells significantly increased TUNEL-positive staining (green fluorescence) compared with the control group. These data demonstrated that 4MOD is able to induce apoptosis of BC cells.

Abnormal autophagy is closely related to the occurrence and development of bladder cancer (Zhu et al., 2019). Salidroside significantly increased the autophagy of T24 cells through the autophagy/PI3K/Akt and MMP-9 signaling induced bladder cancer cells apoptosis (Li T. et al., 2018). Based on this, we explored whether 4MOD affects the progression of bladder cancer cells through autophagy. As shown in Figure 4A, We found that 4MOD significantly enhanced LC3 mRNA expression. LC3, is an autophagic landmark gene, which participates in the formation of the autophagosome (Giménez-Xavier et al., 2009). At the same time, we further identified that 4MOD induced the expression ratio of LC3 (LC3B-II)/LC3B-I and the protein expression of Beclin-1 in J82 and UMUC3 cells in a dose-dependent manner Figure 4B. The statistical analysis of protein quantifications in Figures 4C,D verified it (*p < 0.05). These results suggested that 4MOD induces autophagy of BC cells.

Chloroquine (CQ) first approved for medical use in 1949, have also been widely reported as potential anticancer agents for it can destroy the function of the lysosome and inhibit autophagy (Ferreira et al., 2021). It is one of the classic experiments for observing autophagy flux that the increase in LC3-II protein before and after CQ treatment reflects the amount of autophagosomes degraded by lysosomes. CCK8 assay was utilized to examine the antagonism effect of combining 4MOD with the CQ. As shown in Figures 5A,B, 20 µM 4MOD alone significantly inhibited the proliferation of J82 and UMUC3 cells, while the anti-proliferative effect of 4MOD could be rescued when combined with 5 µM CQ. We next examined the anti-cloning effect of the combination of 4MOD and CQ. Consistent with CCK8 assay, we observed that CQ significantly antagonized the anti-cloning effect of 4MOD (Figures 5C,D). Taken together, these data recognize that inhibition of autophagy may be an effective strategy to increase the anticancer activity of 4MOD in bladder cancer.

Given that Akt/ERK signaling pathways, as critical pathways for tumorigenesis, are key intracellular mediators of cell survival and proliferation signals (Cao et al., 2019). Investigative Akt/ERK signaling during 4MOD treatment are meaningful for us to elaborate its anticancer mechanism. Thus, we explored whether 4MOD induced autophagy by regulating Akt/ERK signaling pathways in the BC cells. As shown in Figures 6A–D, 4MOD diminished the phosphorylation of Akt and/or Erk1/2 while the protein levels of total Erk1/2 and Akt had no significant change in the J82 and UMUC3 cells.

To further determine whether inhibiting Akt/ERK signaling is essential for 4MOD to exert its anti-tumor effect on BC cells, we added Akt specific agonist SC79 in combination with 4MOD in J82 and UMUC3 cells, As shown in Figures 7A,B, 4MOD and SC79 have shown varying degrees of antagonistic effect, the ability of 4MOD to inhibit cancer cell proliferation was reduced after adding SC79. We next observed that when SC79 and 4MOD treated J82 and UMUC3 cells simultaneously, the anti-cloning effect of 4MOD could be reversed (Figures 7C,D). As shown in Figures 7E,F, 4MOD treatment alone inhibited AKT and ERK strongly in both J82 and UMUC3. while SC79 could rescue the 4MOD-inhibited phosphorylation of AKT and ERK when cells were treated with SC79 combination with 4MOD. Meanwhile, the expression ratio of light chain 3-II (LC3-II)/LC3-I were increased in the J82 and UMUC3 cells when the treatment of 4MOD, and SC79 could reverse this phenotype in the UMUC3 while without affecting J82 cells. Figures 7G,H showed the statistical analysis of protein quantifications (*p < 0.05). Together these suggest that 4MOD attenuates BC progression through inhibiting AKT/ERK signaling in vitro.