20(S)-protopanaxadiol (20(S)-PPD), venetoclax, A-1155463 Dihydrochloride, A-1210477, MG132, and cycloheximide were obtained from the Selleck Chemical (Houston, United States).

THP-1 and MOLM-13 cells were provided by Procell Life Science (Wuhan, China), while MV4-11 cells were obtained from ATCC (Manassas, VA, United States). The cell lines were maintained at 37°C in an incubator with 5% CO₂ and RPMI-1640 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS, CLARK Bioscience, United States). The same conditions were used for THP-1 shRNA knockdown cell lines, but puromycin was added at 2 μg/mL.

The Cell Counting Kit-8 (CCK-8; BioSS, Beijing, China) was used to assess cell viability. Following a 48-h exposure of AML cells to varying concentrations of 20(S)-PPD, 10 μL of the CCK-8 reagent was added to each well of a microplate. Subsequently, the cells were incubated for an additional 2 h to allow for the colorimetric reaction to develop. The optical density (OD) at 450 nm of the cells was measured using a microplate reader (BMG LABTECH, GmbH, Germany).

The Annexin V-FITC/propidium iodide (PI) staining (Bestbio, Shanghai, China) was used to determine cell apoptosis. Briefly, a final concentration of 0, 20, 40, 60 μM of 20(S)-PPD was applied to cells plated into 24-well plates. After treating the cells with 20(S)-PPD at different concentrations, a 5-min centrifugation at 1,500 rpm was performed. The cells were then resuspended in 50 μL of 1× binding buffer, and 3 μL of propidium iodide (PI) and 6 μL of Annexin V-FITC solution were added. The mixtures were incubated in the dark for 15 min at room temperature. Flow cytometry analysis (Beckman, United States) was performed after adding 400 μL of binding buffer to each tube. Based on a typical experiment, the average percentages of Annexin V+/PI- (early apoptosis) and Annexin V+/PI+ (late apoptosis or dead cells) were determined.

The JC-10 staining kit (Bestbio, Shanghai, China) was used to assessed mitochondrial membrane potential (MMP). Briefly, AML cells were exposed to 20(S)-PPD at final concentrations of 0, 20, 40, and 60 μM for 8 h. Following treatment, the cells were washed twice with phosphate-buffered saline (PBS) and subsequently centrifuged at 1,500 rpm for 5 min to collect the cells. The cells were then resuspended in 500 μL of JC-10 staining working solution, prepared according to the manufacturer’s instructions, and incubated at 37°C with 5% CO₂ for 30 min. A reduction in MMP is recognized as a hallmark event in the early stages of apoptosis. When the MMP is high, JC-10 preferentially accumulates within the mitochondrial matrix, resulting in the formation of red fluorescence. In contrast, a low MMP leads to the dissociation of JC-10 into monomers, which emit green fluorescence. Subsequently, flow cytometry analysis was conducted to quantify the fluorescence intensity at excitation/emission wavelengths of 510 nm/570 nm for red fluorescence and 488 nm/520 nm for green fluorescence. The ratio of red to green fluorescence intensity serves as an indicator of the cellular MMP, allowing for normalization of the data.

After treating AML cells with 20(S)-PPD at final concentrations ranging from 0 to 60 μM for 8 h, Western blot was performed to detect PARP cleavage and apoptosis-associated proteins. Incubation of cells at 37°C resulted in their harvesting and lysis in ultrasonic lysates containing inhibitors of proteases and phosphatases. The cells were then ultrasonically broken using an ultrasonic crusher with 130 W power, ultrasonication for 1 s, stopping for 1 s, and a total ultrasonication time of 1 min. After centrifuging the total lysate for 10 min at 12,000 rpm, the supernatants were analyzed using a BCA protein assay kit (Thermo Fisher Scientific, United States). We added 20 μg of cell extract to a polyacrylamide-SDS gel containing 5% concentrated gel and 10% separating gel. Once the gels were electrophoretically transferred to PVDF membranes, the membranes were blocked with 5% skim milk for 1 h at room temperature. The membrane, after transfer, was incubated overnight at 4°C with either anti-Bcl-2 family (selleck) or anti-PARP (proteintech) antibodies, both diluted to 1:1,000. After 1 h of incubation with HRP-conjugated secondary antibodies, chemiluminescence (Fine-Do X3, Bioscience, Beijing, China) was used to visualize protein bands using the HRP reaction with its substrates.

Real-time quantitative PCR (qRT-PCR) was used to detect mRNA expression following an 8-h treatment with 20(S)-PPD at concentrations of 0, 20, 40, and 60 μM. Total RNA was isolated using the Total RNA Extraction Kit (Solarbio, China), and cDNA was synthesized using the SureScriptTM First-Strand cDNA Synthesis Kit (GeneCopoeia, China). The MonAmpTM qPCR Kit (Monad Biology, China) and 7,500 Real-Time PCR System (Applied Biosystems, United States) were used in a 20 μL total volume for RT-qPCR. Using the comparative Ct method with β-actin as the housekeeping gene, the 2^−ΔΔCt values were calculated to evaluate mRNA expression levels. The primers used for amplification were as follows: MCL-1:5′-GGAGTTGGTCGGGGAATCTG-3′ and 5′-CTC​CCG​AAG​GTA​CCG​AGA​GA-3′; Bcl-X_L: 5′-AAC​TCT​TCC​GGG​ATG​GGG​TA-3′ and 5′-CTG​CGA​TCC​GAC​TCA​CCA​AT-3′; β-actin: 5′ TCC​TCC​CTG​GAG​AAG​AGC​TAC-3′ and 5′-TCC​TGC​TTG​CTG​ATC​CAC​AT-3′.

Sigma-Aldrich provided the non-target control vector and Bax-/Bak-shRNA constructs, while Dharmacon supplied the Bcl-X_L and MCL-1 open reading frame (ORF) constructs. Lentivirus production was achieved by co-transfecting HEK293T cells with the lentiviral shRNA, pMD-VSV-G, and delta 8.2 plasmids. Viral supernatants were harvested 48 h post-co-transfection, and 1 mL of viral supernatant mixed with 4 μg/mL polybrene (Sigma-Aldrich) was added to THP-1 cells to evaluate infection efficiency. Puromycin at a concentration of 2 μg/mL was used to screen for shRNA lentiviral gene silencing cell lines.

Using the PubChem database (https://pubchem.ncbi.nlm.nih.gov/), we predicted the 3D structure and binding activity of 20(S)-PPD to the relevant targets. The PDB database (https://www.pdb.org/) was used to download the protein structures of the target proteins and remove ligands and non-protein molecules. Before docking could begin, all hydrogens needed to be added to small-molecule drugs and protein structures. With the help of Pymol software, we visualized the docking results using Auto Dock 4. In docking analyses, the model with the smallest G score was regarded as the most appropriate. Docking of 20(S)-PPD with target proteins required binding energies of at least −5.0 kcal/mol. Only results with these energies were accepted.

Statistical analysis was conducted using GraphPad Prism 9.0. Values are presented as mean ± SEM (error bar). Statistical significance was determined with an unpaired two-tailed t-test. The significance threshold was set at P ≤ 0.05 for the column graphs. The IC₅₀ was calculated in non-linear regression by selecting the log (inhibitor) vs normalized response - Variable slope model in the Dose-response-Inhibition category.

AML cells were treated with concentrations ranging from 0 to 60 μM of 20(S)-PPD for 48 h to investigate its anti-proliferative effect. The CCK-8 assay demonstrated that 20(S)-PPD inhibited cell proliferation in a dose-dependent manner (Figure 1A). The IC₅₀ values of 20(S)-PPD for MOLM-13, THP-1, and MV4-11 cells were 29.5 ± 1.4 μM, 44.5 ± 1.5 μM, and 32.5 ± 1.9 μM, respectively (Figure 1B). Further investigation using Annexin V/PI staining and flow cytometry analysis showed that all three tested AML cell lines responded positively to 20(S)-PPD treatment for 8 h, as measured by Annexin V-positive percentage and PARP cleavage (Figures 1C, D). Considering that the decrease in MMP is a hallmark event of early apoptosis, JC-10 staining was performed after treating AML cells with 20(S)-PPD for 8 h to assess the MMP. The results in Figure 1E indicate that 20(S)-PPD reduced MMP in a dose-dependent manner in all tested AML cells. These results demonstrate that AML cells undergo apoptosis in response to 20(S)-PPD. A varying concentration of 20(S)-PPD was then administered for 8 h to peripheral blood mononuclear cells (PBMCs) from healthy donors to evaluate its effect on normal cells. Normal PBMCs treated with 20(S)-PPD did not exhibit an increased percentage of Annexin V-positive cells (Figure 1F), suggesting that 20(S)-PPD may not be toxic to them.

In the mitochondrial apoptotic pathway, Bcl-2 (B Cell Lymphoma 2) proteins Bax and Bak, central effectors of mitochondrial apoptosis, play key roles. When apoptotic signals are initiated, both Bax and Bak insert into the outer mitochondrial membrane, forming oligomers that alter the permeability of the mitochondrial membrane. This change allows the release of cytochrome c, leading to the activation of caspases and ultimately resulting in apoptosis (Shamas-Din et al., 2013). With lentiviral vectors containing Bax/Bak shRNA, we engineered THP-1 cells with a double knockdown of Bax and Bak to determine whether 20(S)-PPD induces apoptosis via the intrinsic/mitochondrial apoptotic pathway. Western blot analysis revealed that, in comparison to the sh-NTC (non-target control), the protein expressions of Bax and Bak were evidently reduced in the Bax/Bak dual knockdown THP-1 cells (Figure 1G). Dual knockdown significantly mitigated 20(S)-PPD-induced apoptosis on AML cells (Figure 1H), indicating that mitochondrial/intrinsic pathways are involved in 20(S)-PPD-induced apoptosis. Based on the data presented in Figure 1, 20(S)-PPD appears to inhibit proliferation and induce apoptosis in AML cell lines, but not in normal PBMCs. The apoptosis induced by 20(S)-PPD appears to be mediated, at least in part, through the intrinsic/mitochondrial apoptotic pathway.

It is well established that the Bcl-2 family proteins are critical regulators of intrinsic/mitochondrial apoptosis, functioning as both promoters and inhibitors of cell death (Czabotar and Garcia-Saez, 2023). Given that 20(S)-PPD-induced apoptosis involves the intrinsic/mitochondrial pathway, we examined the levels of the anti-apoptotic proteins Bcl-2, Bcl-X_L, and MCL-1 and the pro-apoptotic proteins Bax, Bak, and Bim using Western blotting in AML cells following increasing concentrations of 20(S)-PPD for 8 h. The data presented in Figure 2 indicate that treatment with 20(S)-PPD resulted in a reduction of MCL-1 and Bcl-X_L levels, while the expression of Bcl-2, Bfl-1, Bax, and Bak remained unaffected across all three tested AML cell lines. Additionally, the expression of Bim exhibited a slight increase in THP-1 and MV4-11 cells, but remained unchanged in MOLM-13 cells following 20(S)-PPD treatment. These findings suggest that the downregulation of MCL-1 and Bcl-X_L may play a role in mediating 20(S)-PPD-induced apoptosis in AML cells.

Using lentivirus containing ORFs of Bcl-X_L and MCL-1, we successfully overexpressed Bcl-X_L and MCL-1 in THP-1 cells to study their involvement in 20(S)-PPD-induced apoptosis (Figure 3A). Figure 3B shows that the overexpression of Bcl-X_L and MCL-1 partially mitigated 20(S)-PPD-induced apoptosis as determined by annexin V/PI staining and flow cytometry.

A-1210477 (MCL-1 inhibitor) and A-1155463 Dihydrochloride (Bcl-X_L inhibitor), respectively, were co-treated with 20(S)-PPD to further study their roles in 20(S)-PPD-induced apoptosis. As demonstrated in Figures 3C, D, the CCK-8 assay revealed that the inhibitors of MCL-1 or Bcl-X_L synergistically inhibited the proliferation of THP-1 and MOLM-13 cells when combined with 20(S)-PPD. Similarly, the MCL-1 or Bcl-X_L inhibitors significantly enhanced 20(S)-PPD-induced apoptosis in the two AML cell lines, as illustrated in Figures 3E, F. Collectively, these results indicate that 20(S)-PPD induces apoptosis in AML cells by downregulating the levels of MCL-1 or Bcl-X_L, as shown in Figure 3.

20(S)-PPD was treated for 8 h in THP-1 and MOLM-13 cells in order to explore the potential molecular mechanisms by which it regulates Bcl-X_L and MCL-1 expression. qRT-PCR was performed with actin as the reference gene, and the data were subsequently normalized to these baseline values. The relative quantification was exhibited by calculating the 2^−ΔΔCt values. A dose-dependent decrease in Bcl-X_L and MCL-1 mRNA expression was observed in MOLM-13 and THP-1 cells treated with 20(S)-PPD, as shown in Figure 4A.

Subsequently, 20(S)-PPD was investigated for its effect on Bcl-X_L and MCL-1 post-transcriptionally. After treating THP-1 and MOLM-13 cells for 8 h with 20(S)-PPD, 10 μg/mL of Cycloheximide (CHX) was used to inhibit the synthesis of proteins. The expressions of Bcl-X_L and MCL-1 were then measured at various time points using Western blotting to determine the protein degradation rate and half-life. MCL-1 has been demonstrated to be an unstable protein with a half-life ranging from 1.5 to 4 h, in contrast to Bcl-X_L, which exhibits stability with a half-life of approximately 20 h (Rooswinkel et al., 2014). Our data indicated that the half-life of MCL-1 in MOLM-13 cells was 131.5 ± 21.1 min, which decreased to 87.7 ± 17 min following the addition of 20(S)-PPD. Similarly, in THP-1 cells, the half-life of MCL-1 was 133.3 ± 5 min, which further decreased to 73.4 ± 9.6 min after the addition of 20(S)-PPD (Figure 4B). Additionally, the half-life of Bcl-X_L protein was reduced from 48.2 ± 6.6 h to 26.7 ± 5.8 h in MOLM-13 cells and from 36.1 ± 4.6 h to 21.7 ± 2.3 h in THP-1 cells (Figure 4C). These results indicate that 20(S)-PPD accelerated the Bcl-X_L and MCL-1 protein degradation.

To better understand how 20(S)-PPD facilitates the degradation of Bcl-X_L and MCL-1, a virtual molecular docking was conducted using AutoDock 4 software. The docking results revealed that 20(S)-PPD directly interacts with either the MCL-1 or Bcl-X_L proteins, forming hydrogen bonds with ARG-263 on MCL-1, or with PRO-116 on Bcl-X_L (Figures 4D, E). Additionally, 20(S)-PPD exhibited lower Gibbs free energy values of −8.75 kcal/mol and −7.58 kcal/mol for binding to Bcl-X_L and MCL-1, respectively (Table 1). These findings suggest that 20(S)-PPD has a strong binding affinity for both proteins. To further elucidate the role of the proteasome in the degradation of Bcl-X_L and MCL-1 induced by 20(S)-PPD, we examined the impact of the proteasome inhibitor MG132 on the downregulation of these proteins by 20(S)-PPD. As depicted in Figure 4F, the addition of MG132 significantly mitigated the downregulation of Bcl-X_L and MCL-1 by 20(S)-PPD. Collectively, 20(S)-PPD may downregulate MCL-1 or Bcl-X_L protein levels by inhibiting transcription and reducing protein stability.

Venetoclax is a selective and potent oral Bcl-2 inhibitor (Guerra et al., 2019). Venetoclax has been used as monotherapy in patients with relapsed/refractory AML but has demonstrated only modest efficacy with an overall response rate (ORR) of 38% (Liu, 2021; Ong et al., 2022). It appears that AML patients have both intrinsic and acquired resistance to venetoclax, resulting in this low ORR. Indeed, due to venetoclax’s specific targeting of Bcl-2, tumor cells may shift their dependency to other anti-apoptotic proteins such as Bcl-X_L, MCL-1 and Bfl-1 for survival (Shimony et al., 2022). Consequently, this study aims to investigate whether 20(S)-PPD could enhance venetoclax’s sensitivity in treating AML. The CCK-8 assay revealed that the combination of 20(S)-PPD and venetoclax synergistically inhibited THP-1 and MOLM-13 cell proliferation (Figure 5A). Further analysis using Annexin V-FITC/PI staining and flow cytometry indicated that 20(S)-PPD significantly augmented venetoclax-induced apoptosis in THP-1 and MOLM-13 cells (Figure 5B). Additionally, treatment with venetoclax alone (1.25 μM) or in combination with 20(S)-PPD slightly increased the percent of Annexin V-positive cells. However, compared to treatment with venetoclax alone, the combination did not significantly further increase the percent of Annexin V-positive cells (p > 0.05), suggesting that the combination of venetoclax and 20(S)-PPD does not introduce additional toxicity (Figure 5C). Mechanistically, we found that compared to treatment with PPD alone, the combination further downregulated Bcl-X_L and MCL-l, although the level of Bcl-2 remained unchanged (Figure 5D). Collectively, these findings indicate that 20(S)-PPD potentiates the anti-leukemic efficacy of venetoclax in AML cells.