DOI: 10.5061/dryad.d7wm37q9f
title: Targeting autophagy: Polydatin's role in inducing cell death in AML

description: 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.
description: <strong>Cell lines, reagents and
 drug</strong> 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. <strong>Cell
 culture</strong> 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. <strong>Half maximal inhibitory
 concentration (IC50) assay</strong> 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. <strong>CCK-8
 assay</strong> 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%.
 <strong>RT-qPCR assay</strong>
  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. <strong>Cell
 treatment</strong> 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.
 <strong>EDU Staining</strong> 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. <strong>Flow cytometry
 assay</strong> 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. <strong>Western blot
 assay</strong> 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. <strong>ELISA
 assay</strong> 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.
 <strong>Mechanism analysis</strong>
 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. <strong>Animal
 assay</strong> 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).
 <strong>HE Staining</strong> 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. <strong>Molecular
 docking</strong> 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. <strong>Pharmacokinetic
 analysis</strong> 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).
 <strong>Statistical analysis</strong>
 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 &lt; 0.05 was considered significant.
description: # Targeting autophagy: Polydatin's role in inducing cell death in AML
 [https://doi.org/10.5061/dryad.d7wm37q9f](https://doi.org/10.5061/dryad.d7wm37q9f) ## Description of the data and file structure **Fig.4 Polydatin activates ATG5-mediated autophagy pathway in vitro**  Molecular docking diagram of polydatin and ATG5; ## Code/software Autodock 1.5.6