Acute myeloid leukemia (AML), a malignant disorder of the hematopoietic system, arises from leukemic stem cells (LSCs) and is the most prevalent form of blood cancer in adults.  This study aimed to evaluate the therapeutic potential of polydatin (PD) in AML through ex vivo and in vivo studies, respectively. This study was prompted by PD's novel role in enhancing tumor apoptosis and modulating autophagy.  In vitro studies were conducted using the PD-responsive AML cell line KASUMI-1 and  found that PD was able to suppress cell proliferation and induce apoptosis by regulating the autophagy pathway. Subsequently, molecular docking was employed to predict the interaction between PD and Autophagy-related protein 5 (ATG5), a key regulator in the autophagy pathway. It was observed that PD inhibited the ubiquitination of ATG5 and enhanced its protein stability, leading to an increase in ATG5 protein levels and subsequent activation of the autophagy pathway (see in Abstract Graphed). The effectiveness and safety of PD in treating AML were confirmed through in vivo experiments using a mouse transplant tumor model, yielding definitive results. Collectively, these results suggest that PD is a promising candidate for the early therapeutic intervention of AML, with a strong potential for clinical application.

Cell lines, reagents and drug

Human AML cells: KG-1(No.CCL-246.1), HL-60 (No.CRL-3306), KASUMI-1 (No.CRL-2724), KASUMI-6 (No.CRL-2775), murine AML cell lines: C1498 (No.TIB-49), all purchased from American Type Culture Collection (ATCC) Cell Bank, USA. SPF NCG mice, 4-5 weeks old, weighing 16-18 g, were purchased from Beijing Vital River Laboratory Animal Co., Ltd. Animal production license number: SCXK (Beijing) 2021-0011.

Reagents and drugs: fetal bovine serum (No.A5669701), RPMI1640 medium (No.11875119) were purchased from Gibco, USA; penicillin-streptomycin solution (No.C0222), cell counting kit-8 (No.C0038) and BCA protein concentration quantitative kit (No.P0010S) were purchased from Shanghai Beyotime Biotechnology. Hematoxylin-eosin (HE) staining kit (No. G1120) was purchased from Soleibao Biotechnology Company, Beijing, China. 5-bromo-2-deoxyuracil (EDU) kit (No. KGA9602-100), human interleukin-6 (IL-6), IL-1β, Tumor necrosis factor -α (TNF-α) enzyme-linked immunosorbent assay (ELISA) kit (No. KGC1111-48, KGC1103-48, KGC1122-48), flow cytometry Annexin-v-FITC kit (No. KGA1101-100) were purchased from China Jiangsu Keygen Biological Co., Ltd.;

Mouse hemoglobin (Hb) ELISA kit (No. LE-M1587) was purchased from Hefei Lyle Biotechnology Co., Ltd., China, and mouse erythropoietin (Epo) ELISA kit (No.XG-E989530) was purchased from Shanghai Xige Biotechnology Co., Ltd., China. human B-cell lymphoma-2 (BCL2) antibody (No.12789-1-AP), human Bcl-2 Associated X protein (BAX) antibody (No. 50599-2-Ig), human Poly ADP-ribose polymerase (PARP) antibody (No.13371-1-AP) , ATG5 Monoclonal antibody (No. 66744-1-Ig) and human Cleaved caspase3 antibody (No.19677-1-AP) were purchased from Wuhan Proteintech Biological Co., Ltd. Polydatin (No.HY-N0120A) was purchased from MCE Biological Co., Ltd. The purity of Polydatin (batch number: HY-N0120A-29) was 99.57%.

Instruments: DILITCEN22 desktop centrifuge, Suzhou Beirui Instrument Co., Ltd, China; hBS-ScanX full wavelength microplate reader, Nanjing Detieer Experimental Equipment Co., Ltd, China; dYJ-905 inverted metallographic microscope, Shanghai Dianying Optical Instrument Co., Ltd, China; Invitrogen iBright all-round gel imager, Thermo Fisher Science and Technology Co., Ltd, USA; FACSCanto II flow cytometer, BD Biomedical, USA; 1290 LC-MS, 1290 UHPLC with 6230TOF MS System, Agilent, USA.

Cell culture

The cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin at 37 °C with 5% CO2. Upon reaching a cell density exceeding 80%, the cells were harvested by centrifugation and passaged at a ratio of 1:3.

Half maximal inhibitory concentration (IC50) assay

KG-1, HL-60, C1498, KASUMI-1, and KASUMI-6 cells were plated in 96-well plates at a density of 8 × 103 cells per well. PD stock solution was prepared by dissolving in dimethyl sulfoxide (DMSO) according to the manufacturer's instruction. Following a 12-hour incubation period to allow for cell adherence, the PD stock solution was diluted with culture medium to achieve  working concentrations of 1, 5, 10, 20, 40, and 100 μM. The cells were then exposed to these varying concentrations of PD for 48 hours(Li et al., 2017).  Subsequently, 10 μL of cell counting kit 8 (CCK-8) solution was added to each well, followed by a 2-hour incubation in darkness. Absorbance at a wavelength of 450 nm was measured using an enzyme labeling instrument, and the IC50 value was calculated using GraphPad Prism software.

CCK-8 assay

The KG-1, HL-60, C1498, KASUMI-1, and KASUMI-6 cells were seeded in 96-well plates at a density of 3 × 10³ per well. After 12 hours of adherence, the cells were treated with PD (0, 10, 20, and 40 μM), respectively, with 5 replicate wells in each group. At 24, 48, 72, and 96 hours after drug treatment, 10 μL of CCK-8 solution was added to each well, which was then incubated in the dark for 2 hours. The absorbance was then measured at a wavelength of 450 nm. The cell proliferation rate was calculated according to the following formula: The cell proliferation rate is calculated as follows: (Absorbance value of experimental group - Absorbance value of blank well) / (Absorbance value of control group - Absorbance value of blank well) × 100%.

RT-qPCR assay

 KG-1, HL-60, C1498, KASUMI-1 and KASUMI-6 were seeded in 6-well plates at a density of 1 × 106 per well. After adherent for 12 h, the cells were treated with PD (0, 10, 20, and 40 μM), respectively. After 48 h, the total RNA was extracted with Trizol. cDNA was synthesized using a reverse transcription kit according to the manufacturer's instructions. RT-qPCR was then conducted with a SYBR Prime Script RTPCR kit utilizing custom primers (in Supplementary Table S1). The relative expression levels of mRNA were quantified using the 2^-ΔΔCt method.

Cell treatment

KASUMI-1 cells were seeded in 96-well plates (or 6-well plates) at a density of 3 × 103 (or 1 × 106) per well. After 12 h of attachment, the cells were treated with PD (0, 10, 20, and 40 μM) for 48 h, respectively. After treatment, the relevant experimental tests were performed. The CCK-8 experimental steps were performed according to the above description.

EDU Staining

The EDU assay was carried out according to the protocol from the EDU kit manual after treatment. In summary, the EDU reagent was diluted with culture medium to create a working solution, and then was added to the cells for incubation for 6 h. After the incubation, 4% paraformaldehyde solution was added to fix the cells, and 1 % Triton X-100 solution was used for membrane penetration. The Click-iT EdU reaction reagent was prepared according to the EDU kit manual and added to the cell culture plate for further incubation for 30 min. Finally, the cells were observed under a fluorescence inverted microscope, and photographs were taken for record.

Flow cytometry assay

Following drug treatment, the procedures outlined in the Annexin V/PI Apoptosis Detection Kit manual were adhered to. This included the collection and washing of cells and supernatant with PBS solution three times, followed by the addition of 5 μL of Annexin V solution and 5 μL of PI solution. Subsequently, the samples were thoroughly mixed and incubated at room temperature in the absence of light for 15-30 minutes. The samples were then analyzed using appropriate equipment, and the resulting data were processed utilizing Flow jo10.0 software.

Western blot assay

The total protein of the cells was extracted and quantified using a BCA kit after treatment. Subsequently, according to the sample order, 40 μg samples were fractionated using 10% SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently transferred onto a PVDF membrane. Following blocking with 5% non-fat milk, the membrane was incubated overnight at 4°C with the respective primary antibody solution, which was diluted at a ratio of 1:1000. This was followed by a 5-hour incubation with the appropriate secondary HRP-conjugated antibodies (1:8000). Ultimately, the protein signal was visualized using an ECL detection kit, and the strip's gray value was analyzed using Image J software.

ELISA assay

After the cells were treated with drugs, the operation was performed according to the instructions of the ELISA detection kit. In short, the cell supernatant solution was collected and added to the pre-coated well plate. After the incubation, the antibody labeled with the enzyme was washed and added to continue the incubation with the specific antibody. The color reaction was performed, and the absorbance was measured by a microplate reader.

Mechanism analysis

In terms of mechanism analysis, the cells were divided into 0 μM group, 20 μM PD group, ATG5 autophagy inhibitor (agent-82,10 μM) group, and 20 μM PD + ATG5 autophagy inhibitor combination group (agent-82,10 μM) group. After 48 h of treatment, the cells were tested according to the CCK-8, EDU, ELISA, Western blot and flow cytometry operation steps described above.

Animal assay

NCG mice used to construct KASUMI-1 subcutaneous transplantation tumor model. All mice were housed in a sterile mouse house environment, alternating day and night every 12 hours, and were given adequate food and drinking water. After one week of adaptation, the AML model was established by subcutaneous injection of KASUMI-1 cells (2 × 107) in the right limb of mice. When the tumor volume reached 100 mm3, the mice were treated with drugs.  PD stock solution was dissolved in 0.9% saline solution to prepare the treatment solutions. The mice in each experimental group received the PD solution via intragastric gavage once daily at dosages of 50 mg/kg, 100 mg/kg, and 200 mg/kg, respectively. The control group was given an equal amount of  DMSO diluted in saline solution(Zhang et al., 2019, Li et al., 2023). During the treatment, the tumor volume and the body weight of the mice were measured every 3 days. The tumor volume was calculated according to the formula V = L × W2/2, where L represents the tumor length and W represents the tumor width. After 4 weeks, peripheral blood was extracted from the tail vein of mice, and 2 mL was mixed with 38 mL Turk blood diluent, and then the white blood cell count (WBC) was observed under a microscope. The remaining blood was centrifuged at 12,000 rpm for 5 min, and the supernatant was collected. The expression levels of hemoglobin (Hb) and erythropoietin (Epo) were detected using a whole blood automatic analyzer. After that, the mice were euthanized, and the lung, kidney, spleen and liver tissues of the mice were collected, and then the tissue samples were stained with hematoxylin and eosin. All experimental animals were euthanized for cervical dislocation with a high concentration of CO2, and the animal experiments were approved by the Ethics Committee for the Institutional Animal Care and Ethics Committee of The Second Affiliated Hospital, University of South China. (Animal Ethics 230319).

HE Staining

After the lung, kidney, spleen and liver tissues of mice were fixed and embedded, they were cut into 6 μm slices by frozen section machine. The operation was carried out according to the instructions of HE staining kit, and the pathological structure of the tissues was analyzed by taking photos under the microscope.

Molecular docking

The three-dimensional structure of PD and ATG5 protein was obtained by PubChem. The active binding sites of PD and ATG5 were defined by AutoDock software, and the network was generated.

Pharmacokinetic analysis

The real-time drug concentration of PD in the blood of healthy mice was analyzed by high-performance liquid chromatography (HPLC), with six mice in each group undergoing alternate blood collection. The blood was collected at the following time points: 0 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, and 24 h. The following parameters were used for the determination: flow rate: The flow rate was set at 0.1ml/min, the temperature at 25°C, the column at Bonus-PR, and the mobile phase comprised 20mM sodium phosphate buffer (pH2.5)-acetonitrile (90:10).

Statistical analysis

All experiments were repeated more than 3 times, and data analysis was performed using GraphPad Prism (version 9.0). All results are presented as mean ± standard error (SEM). The difference between the two groups was performed using a 2-tailed Student's t-test. One-way analysis of variance (ANOVA) was used for comparison between multiple groups. P < 0.05 was considered significant.