The antitumor effects and apoptotic mechanism of 20(S)-Protopanaxadiol in acute myeloid leukemia.

AML patients’ samples were obtained from the First Hospital of Jilin University, Changchun, China. PBMCs were donated by healthy individuals. This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of The 10.13039/501100017585 First Hospital of Jilin University .

This work was supported by Jilin Tianhua Health Charity Foundation ( J2023JKJ021 ) and Sanya Science and Technology Innovation Special Project ( 2022KJCX32 ).

We initially examined the effects of 20(S)-PPD on the proliferation of AML cells using MTT assays in six AML cell lines, including MOLM-13, MV4-11, HL-60, NB4, CTS, and THP-1. Cells were treated with various concentrations of 20(S)-PPD for 48 h. The results showed that the reduction in viable cells was dose-dependent ( Fig. 1 A), with IC50 values ranging from 20 to 40 μM ( Fig. 1 B). To further determine whether 20(S)-PPD could induce apoptosis in AML cells, MOLM-13, MV4-11, and HL-60 cells were treated with 20–30 μM 20(S)-PPD for 24 h. Flow cytometry analysis revealed a dose-dependent induction of apoptosis by 20(S)-PPD ( Fig. 1 C), which was further confirmed by the cleavage of PARP ( Fig. 1 D). Fig. 1 20 (S) -PPD suppresses proliferation and induces apoptosis in AML cells. (A) The MTT assay was performed at 48 h following treatment with 0–40 μM 20(S)-PPD in AML cell lines. (B) The IC50 values of 20(S)-PPD were determined. Annexin V-FITC/PI staining and flow cytometry (C) or Western blotting (D) was performed at 24 h after treatment with variable 20(S)-PPD in MOLM-13, MV4-11, and HL-60 cells. Primary AML patient samples (E) or normal PBMC (F) were treated with the indicated 20(S)-PPD for 24 h and then analyzed by Annexin V-FITC/PI staining and flow cytometry. ∗, p < 0.05 compared to vehicle control. Fig. 1 20 (S) -PPD suppresses proliferation and induces apoptosis in AML cells. (A) The MTT assay was performed at 48 h following treatment with 0–40 μM 20(S)-PPD in AML cell lines. (B) The IC50 values of 20(S)-PPD were determined. Annexin V-FITC/PI staining and flow cytometry (C) or Western blotting (D) was performed at 24 h after treatment with variable 20(S)-PPD in MOLM-13, MV4-11, and HL-60 cells. Primary AML patient samples (E) or normal PBMC (F) were treated with the indicated 20(S)-PPD for 24 h and then analyzed by Annexin V-FITC/PI staining and flow cytometry. ∗, p < 0.05 compared to vehicle control. Furthermore, we evaluated the effects of 20(S)-PPD on primary AML patient samples and found that it could also induce apoptosis in these samples ( Fig. 1 E). To ensure the safety of 20(S)-PPD, we investigated its impact on normal peripheral blood cells and discovered that even at elevated concentrations, 20(S)-PPD did not significantly induce apoptosis in these cells ( Fig. 1 F), suggesting its potential safety for clinical application. To explore the specific mechanism underlying 20(S)-PPD-mediated apoptosis in AML cells, we identified 973 AML-related genes from the GeneCards and OMIM database with a correlation score of >16. We then used the SwissTargetPrediction database to predict 78 potential targets of 20(S)-PPD. Subsequently, by intersecting these 78 predicted targets and the 973 AML-related genes, we obtained 22 research targets, including MET, ALK, PIK3CB, MAPK10, MTOR, MAPK14, KIT, F2 PTGS2, ESR1, PIK3CD, HSP90AA1, KDR, PIK3CG, MDM2, PIK3CA, MAPK8, IL6ST, ACP1, REN, AR, and CYP3A. Then, these 22 genes were analyzed with the DAVID platform for KEGG pathway enrichment, revealing significant enrichment in pathways such as PI3K/AKT, Ras and other pathways ( Fig. 2 A). Fig. 2 20 (S) -PPD activates the PERK/ATF4/CHOP pathway and inhibits the PI3K/AKT/mTOR pathways, while up-regulates p-ERK1/2 in AML cells. (A) KEGG pathway enrichment analysis of the overlapping genes. (B) Flow cytometry and Western blotting were conducted following 20(S)-PPD treatment in MOLM-13 and MV4-11 cells. (C–E) Cells were treated with 20(S)-PPD alone or in combination with 2 μM PI3K/mTOR inhibitor VS-5584 (C), 500 nM ERK1/2 inhibitor SCH772984 (D), or 500 nM PERK inhibitor ISRIB (E) for the indicated durations, followed by flow cytometry or Western blotting. ∗, P < 0.05 from vehicle control. #, P < 0.05 from individual drug. Fig. 2 20 (S) -PPD activates the PERK/ATF4/CHOP pathway and inhibits the PI3K/AKT/mTOR pathways, while up-regulates p-ERK1/2 in AML cells. (A) KEGG pathway enrichment analysis of the overlapping genes. (B) Flow cytometry and Western blotting were conducted following 20(S)-PPD treatment in MOLM-13 and MV4-11 cells. (C–E) Cells were treated with 20(S)-PPD alone or in combination with 2 μM PI3K/mTOR inhibitor VS-5584 (C), 500 nM ERK1/2 inhibitor SCH772984 (D), or 500 nM PERK inhibitor ISRIB (E) for the indicated durations, followed by flow cytometry or Western blotting. ∗, P < 0.05 from vehicle control. #, P < 0.05 from individual drug. To determine the timing of 20(S)-PPD-induced apoptosis, variable concentrations of 20(S)-PPD were administered at the indicated intervals. Apoptosis was observed as early as 6 h post-treatment, with a more pronounced effect at 24 h ( Fig. 2 B). Accordingly, protein levels were primarily analyzed at these two time points. Given that KEGG enrichment analysis indicated that 20(S)-PPD affects the PI3K/AKT and Ras pathways in AML cells, we examined the impact of 20(S)-PPD on key proteins in these pathways. As shown in Figs. 2 C and 20(S)-PPD decreased the levels of p-AKT (Ser473) and p-S6, while total AKT levels remained unchanged. We also found that the PI3K/mTOR dual inhibitor VS-5584 further reduced p-AKT (Ser473) levels and enhanced 20(S)-PPD-induced apoptosis ( Fig. 2 C). These results demonstrate that 20(S)-PPD-induced apoptosis is associated with the PI3K/AKT/mTOR pathway. Surprisingly, 20(S)-PPD increased p-ERK1/2 protein levels in AML cells while it did not change total ERK1/2 protein levels ( Fig. 2 D). In addition, the ERK1/2 inhibitor SCH772984 attenuated the 20(S)-PPD-induced elevated p-ERK1/2 protein levels, while further enhancing the pro-apoptotic effects of 20(S)-PPD ( Fig. 2 D). This suggests that upregulation of p-ERK1/2 may serve as a potential resistance mechanism against the anti-tumor effects of 20(S)-PPD. We also examined the PERK/ATF4/CHOP pathway, which is known to play a crucial role in 20(S)-PPD-induced apoptosis [ 20 , 21 ]. Therefore, we detected the changes of p-eIF2α and ATF4 protein levels in MOLM-13 and MV4-11 cells. We found that 20(S)-PPD increased p-eIF2α and ATF4 protein levels ( Fig. 2 E). Moreover, the PERK-selective inhibitor ISRIB counteracted the 20(S)-PPD-induced upregulation of p-eIF2α protein levels and decreased apoptosis induced by 20(S)-PPD ( Fig. 2 E). In summary, our results demonstrate that 20(S)-PPD inhibits the PI3K/AKT/mTOR pathway, activates the PERK/ATF4/CHOP pathway, and upregulates p-ERK1/2 in AML cells. To determine whether 20(S)-PPD induces apoptosis via the intrinsic pathway, Bax and Bak were knocked down in MV4-11 cells. The results showed that dual knockdown of Bax and Bak attenuated 20(S)-PPD-induced apoptosis ( Fig. 3 A), indicating that 20(S)-PPD-induced apoptosis was partially through intrinsic pathway. Studies have shown that alterations in the AKT or ERK pathways can affect downstream proteins such as members of the Bcl-2 family [ 22 ]. Bcl-2 family proteins mainly trigger the intrinsic apoptosis pathway by modulating the permeability of the mitochondrial outer membrane, which is mainly caused by the homodimerization of Bax or Bak, leading to membrane perforation [ 23 ]. We then tested the effect of 20(S)-PPD on Bcl-2 family proteins and found that it reduced Mcl-1 protein levels in AML cells, while other proteins remained unchanged ( Fig. 3 B). Fig. 3 c-Myc mediates the pro-apoptotic effect of 20(S)-PPD in AML cells. (A) MV4-11 cells with Bak and Bax double knockdown were treated with 20(S)-PPD for 24 h, followed by Annexin V-FITC/PI staining and flow cytometry analysis. (B–C) MOLM-13 and MV4-11 cells were treated with 20(S)-PPD for 6 h or 24 h and then analyzed by Western blotting. (D–E) Cells were treated with 20(S)-PPD alone or in combination with the pan-caspase inhibitor Z-VAD-FMK (D) or 50 μM c-Myc inhibitor 10058-F4 (E) for 24 h, followed by flow cytometry or Western blotting. ∗, P < 0.05 from vehicle control. #, P < 0.05 from individual drug. Fig. 3 c-Myc mediates the pro-apoptotic effect of 20(S)-PPD in AML cells. (A) MV4-11 cells with Bak and Bax double knockdown were treated with 20(S)-PPD for 24 h, followed by Annexin V-FITC/PI staining and flow cytometry analysis. (B–C) MOLM-13 and MV4-11 cells were treated with 20(S)-PPD for 6 h or 24 h and then analyzed by Western blotting. (D–E) Cells were treated with 20(S)-PPD alone or in combination with the pan-caspase inhibitor Z-VAD-FMK (D) or 50 μM c-Myc inhibitor 10058-F4 (E) for 24 h, followed by flow cytometry or Western blotting. ∗, P < 0.05 from vehicle control. #, P < 0.05 from individual drug. Studies have shown that the PI3K/AKT pathway regulates multiple genes involved in cell survival and proliferation, including the oncogene c-Myc [ 22 ]. Given this, we next assessed c-Myc levels in AML cells. Western blotting results showed that c-Myc protein levels were significantly reduced following 20(S)-PPD treatment ( Fig. 3 C). We then introduced the pan-caspase inhibitor Z-VAD-FMK to inhibit caspase activity and block apoptosis in AML cells. Flow cytometry results showed that Z-VAD-FMK could attenuate 20(S)-PPD-induced apoptosis ( Fig. 3 D). Notably, compared to 20(S)-PPD alone, Mcl-1 protein levels were up-regulated, while c-Myc protein levels remained largely unchanged following the combination of 20(S)-PPD and Z-VAD-FMK ( Fig. 3 D). These findings suggest that Mcl-1 levels are modulated by apoptotic processes, whereas c-Myc levels remain unaffected by apoptosis, suggesting that c-Myc may be a key mediator of 20(S)-PPD-induced apoptosis. Subsequently, we combined 20(S)-PPD with the c-Myc inhibitor 10058-F4 and found that 10058-F4 potentiated the inhibitory effect of 20(S)-PPD on c-Myc protein levels and further increased 20(S)-PPD-induced apoptosis ( Fig. 3 E). These results demonstrate that c-Myc mediates the pro-apoptotic effect of 20(S)-PPD in AML cells. To explore the mechanism by which 20(S)-PPD downregulates c-Myc, we first examined its effect on c-Myc mRNA levels. RT-PCR analysis showed that 20(S)-PPD significantly reduced c-Myc mRNA levels in MOLM-13 and MV4-11 cells ( Fig. 4 A). We then assessed the impact of 20(S)-PPD on c-Myc mRNA stability and found that 20(S)-PPD significantly accelerated the degradation rate of c-Myc mRNA ( Fig. 4 B), suggesting that 20(S)-PPD destabilizes c-Myc mRNA. Additionally, we investigated whether 20(S)-PPD affects c-Myc protein stability. Western blotting analysis showed that 20(S)-PPD did not alter the degradation rate of c-Myc protein ( Fig. 4 C), indicating that 20(S)-PPD does not affect c-Myc protein stability. Fig. 4 20 (S) -PPD reduces c-Myc mRNA levels and mRNA stability. (A) Cells were treated with variable 20(S)-PPD for 6 h and analyzed by RT-PCR. (B–C) Cells were treated with 20(S)-PPD alone or in combination with 5 μM actinomycin D and analyzed by qRT-PCR (B), or with 10 μg/mL cycloheximide and then followed by Western blotting (C). ∗, P < 0.05 from vehicle control. Fig. 4 20 (S) -PPD reduces c-Myc mRNA levels and mRNA stability. (A) Cells were treated with variable 20(S)-PPD for 6 h and analyzed by RT-PCR. (B–C) Cells were treated with 20(S)-PPD alone or in combination with 5 μM actinomycin D and analyzed by qRT-PCR (B), or with 10 μg/mL cycloheximide and then followed by Western blotting (C). ∗, P < 0.05 from vehicle control. We have shown that c-Myc mediates the pro-apoptotic effect of 20(S)-PPD in AML cells, although 20(S)-PPD does not affect Bcl-2 protein levels. Studies have demonstrated that sustained overexpression of Bcl-2 is essential for c-Myc-mediated tumorigenesis in mice [ 24 ]. Therefore, combining drugs that target c-Myc with Bcl-2 inhibitors may represent a promising strategy for improving AML treatment. Given that 20(S)-PPD downregulates c-Myc expression in AML, combining it with ABT-199, a highly selective oral Bcl-2 inhibitor, could potentially enhance the anti-tumor activity of ABT-199. We first evaluated the combined effects of these two agents on cell proliferation and apoptosis in AML. Compared to individual treatments, the combination significantly inhibited cell proliferation with a CI value of <1 ( Fig. 5 A and B), indicating a synergistic anti-tumor effect of these two drugs. The combination also increased apoptosis ( Fig. 5 C) and significantly reduced c-Myc mRNA and protein levels in AML cells ( Fig. 5 D and E). To further assess whether c-Myc is functionally involved in the synergistic effect of 20(S)-PPD and ABT-199, we performed c-Myc knockdown (KD) in AML cell. The results showed that c-Myc knockdown led to increased apoptosis in AML cells. Moreover, compared to normal AML cells, c-Myc-KD AML cells exhibited a greater increase in apoptosis upon treatment with 20(S)-PPD and ABT-199 ( Fig. 5 F). These findings demonstrate that the combination of 20(S)-PPD and ABT-199 can lead to synergistic anti-tumor effect in AML cells. Fig. 5 20 (S) -PPD enhances the anti-tumor effect of ABT-199 in AML cells . (A) The MTT assay was performed 24 h after treatment with 20(S)-PPD or ABT-199. (B) CI values for 20(S)-PPD and ABT-199 in MOLM-13 and MV4-11 cells. (C–E) MOLM-13 and MV4-11 cells were treated with 20(S)-PPD or ABT-199 alone or in combination for 6 h, followed by flow cytometry (C), qRT-PCR (D), or Western blotting (E). (F) Lentivirus-mediated c-Myc knockdown (KD) was performed, with non-target control (NTC) as the negative control, and validated by Western blotting. Control or KD cells were treated with the indicated drugs for 6 h, followed by flow cytometry analysis. ∗, P < 0.05 from vehicle control. #, P < 0.05 from individual drug. Fig. 5 20 (S) -PPD enhances the anti-tumor effect of ABT-199 in AML cells . (A) The MTT assay was performed 24 h after treatment with 20(S)-PPD or ABT-199. (B) CI values for 20(S)-PPD and ABT-199 in MOLM-13 and MV4-11 cells. (C–E) MOLM-13 and MV4-11 cells were treated with 20(S)-PPD or ABT-199 alone or in combination for 6 h, followed by flow cytometry (C), qRT-PCR (D), or Western blotting (E). (F) Lentivirus-mediated c-Myc knockdown (KD) was performed, with non-target control (NTC) as the negative control, and validated by Western blotting. Control or KD cells were treated with the indicated drugs for 6 h, followed by flow cytometry analysis. ∗, P < 0.05 from vehicle control. #, P < 0.05 from individual drug. Cytarabine (AraC) is still a widely used drug in AML treatment. However, drug resistance frequently results in high relapse rates. The recurrence rate is approximately 50 % in younger patients (60 years old) [ 25 ]. This highlights the urgent need for new strategies to overcome AraC resistance and improve outcomes in relapsed AML patients. Studies have shown that c-Myc protein levels are significantly upregulated in AraC-resistant (AraC-R) AML cells [ 16 ], suggesting that inhibiting c-Myc could help overcome AraC resistance in AML patients. In AML parental cells, 20(S)-PPD and ABT-199 significantly reduced c-Myc mRNA and protein levels. We then explored whether this combination could exhibit anti-tumor activity in AraC-R AML cells. In MV4-11/AraC-R cells, compared with the individual treatment, the combination can further inhibit cell proliferation with a CI value of <1( Fig. 6 A), indicating a synergistic anti-tumor effect in MV4-11/AraC-R cells. There was also an observed increase in cell apoptosis (( Fig. 6 B). Moreover, the combined treatment could also significantly reduce c-Myc protein levels and mRNA levels in AraC-R AML cell ( Fig. 6 C and D). We also performed c-Myc knockdown in AraC-R AML cells, and the results were consistent with those observed in normal AML cells. The combination of 20(S)-PPD and ABT-199 induced more apoptosis in c-Myc-KD AraC-R cells than that in AraC-R AML cells. ( Fig. 6 E). These findings suggest that the combination of 20(S)-PPD and ABT-199 elicits a synergistic anti-tumor effect in AraC-R AML cells. Fig. 6 Synergistic antitumor effects of 20(S)-PPD and ABT-199 in AraC-resistant (AraC - R) cells. (A) The MTT assay was conducted 48 h post-treatment with 20(S)-PPD or ABT-199 in MV4-11/AraC-R cells, with the figure illustrating the CI values for the two drugs. (B–D) Cells were treated with 20(S)-PPD or ABT-199 alone or in combination for 6 h, followed by flow cytometry(B), qRT-PCR (C), or Western blotting (D). (E) Western blotting was used to validate c-Myc knockdown in MV4-11/AraC-R cells. Both control and KD cells were treated with the indicated drugs for 6 h, and then analyzed by flow cytometry. ∗, P < 0.05 from vehicle control. #, P < 0.05 from individual drug. Fig. 6 Synergistic antitumor effects of 20(S)-PPD and ABT-199 in AraC-resistant (AraC - R) cells. (A) The MTT assay was conducted 48 h post-treatment with 20(S)-PPD or ABT-199 in MV4-11/AraC-R cells, with the figure illustrating the CI values for the two drugs. (B–D) Cells were treated with 20(S)-PPD or ABT-199 alone or in combination for 6 h, followed by flow cytometry(B), qRT-PCR (C), or Western blotting (D). (E) Western blotting was used to validate c-Myc knockdown in MV4-11/AraC-R cells. Both control and KD cells were treated with the indicated drugs for 6 h, and then analyzed by flow cytometry. ∗, P < 0.05 from vehicle control. #, P < 0.05 from individual drug.

MOLM-13 was purchased from AddexBio (San Diego, USA). MV4-11, THP-1, HL-60 were purchased from ATCC (Manassas, USA), NB-4 were purchased from DSMZ (Braunschweig, Germany), CTS was provided by Dr. A. Fuse (National Institute of Infectious Diseases, Tokyo, Japan). All the cells were cultured as previously described [ 16 ]. AraC-resistent MV4-11 cells were co-cultured with 1.1 μM AraC to maintain AraC resistance. 20(S)-PPD was obtained from Yuanye (Shanghai, China). ISRIB, SCH772984, Z-VAD-FMK, VS-5584, 10058-F4, and ABT-199 were obtained from AbMole. Samples from AML patients were collected at the First Hospital of Jilin University. Normal peripheral blood mononuclear cells (PBMC) were donated by healthy volunteers. Detailed information on the AML patients was provided in Supplementary Table 1 . AML cells were suspended in culture medium containing varying concentrations of 20(S)-PPD, ABT-199, either alone or in combination. After incubation at 37 °C for 44 h, MTT (final concentration 1 mM) was added to each well. After another 4 h, 100 μL lysis was introduced, and the absorbance was measured at 590 nm using a plate reader. AML cells were processed with the specified drugs. Cells were stained with fluorescein isothiocyanate (FITC) -conjugated Annexin V and propidium iodide (PI) and assayed by flow cytometer. The ratio of Annexin V-positive cells indicates the proportion of apoptotic cells, with Annexin V+/PI- representing early apoptosis while Annexin V+/PI + reflecting late apoptosis or dead cells. Western blots were performed as previously described [ 17 ]. Briefly, Cells were lysed by sonication in the 10 mM Tris-Cl containing 1 % SDS, supplemented with protease and phosphatase inhibitors (Roche Diagnostics). Total protein concentration was measured using the BCA method. Cell lysates were then subjected to SDS-PAGE, transferred to PVDF membranes, and probed with the indicated antibodies for immunoblotting. The following antibodies were used: anti-cleaved-PARP (13371-1-AP), -ERK (16443-1-AP), -Bax (50599-2-Ig), -Mcl-1 (16225-1-AP), -β-actin (66009-1-Ig) (Proteintech, Rosemont, IL, USA), -p-eIF2S1(S51)(A5941), -ATF4(A5514), -AKT (A5031), -p-ERK(T202/Y204; A5036), -Bak (A5068), -Bim (A5114), -Bcl-xL (A5091), -c-Myc(A5011) (Selleck Chemicals, Shanghai, China), and -p-AKT (S473; 3787S), -p-S6(5364S) (Cell Signaling Technologies, Danvers, USA) antibodies. Membranes were scanned and analyzed with the Odyssey infrared scanner (LI-COR). The qRT-PCR analysis was carried out as described [ 18 ]. The primer sequences used were as follows: c-Myc forward (5'-GTGGTCTTCCCCTACCCTCT-3') and reverse (5'-CGAGGAGAGCAGAGAATCCG-3'); 36B4 forward (5'-CGACCTGGAAGTCCAACTAC-3') and reverse (5'-ATCTGCTGCATCTGCTTG-3'). AML-related genes were gathered from the GeneCards database ( https://www.genecards.org/ ) and OMIM database ( https://www.omim.org/ ), based on the correlation score (>16). SwissTargetPrediction ( http://www.swisstargetprediction.ch/ ) was used to predict potential target genes of 20(S)-PPD. The intersection of these predicted targets and AML-related genes was then submitted to the DAVID database for KEGG signaling pathway enrichment analysis under the functional annotation category. The resulting data were further processed using the Micro-Informatics Online Drawing Website to generate the diagram. The lentiviral constructs carrying c-Myc shRNA were obtained from Sigma. Lentiviral particles were produced according to established protocols [ 19 ]. Briefly, HEK-293T cells were co-transfected lentivirus construct, pMD-VSV-G envelope plasmid, and delta 8.2 packaging plasmid using Polyethylenimine (Polysciences). Lentivirus-containing medium was collected on Day 3 post-transfection and then concentrated using the Lenti-X Concentrator (Clontech). AML cells were transduced with the concentrated lentivirus in normal growth media. At 48 h after transduction, the cells were treated with the indicated drug, followed by Western blotting analysis and assessment of apoptosis. Student's two-tailed t -test was used to determine the difference between the means of the two groups. p < 0.05 was defined as a significant difference. Data are expressed as mean ± SD. Drug interactions were analyzed using the Calcusyn software (Biosoft), which computes combination index (CI) values according to the median-effect principle. A CI value of 1 indicates synergy, additivity, or antagonism, respectively.

Ginsenosides, the primary bioactive components of ginseng plants, have shown significant anti-tumor effects in various cancer cells. Notably, 20(S)-PPD stands out as a particularly effective compound within the ginsenoside family for its anti-tumor activity [ 10 ]. For instance, 20(S)-PPD has been shown to inhibit tumor growth by targeting the PI3K/AKT/mTOR pathway in breast cancer [ 12 ]. Furthermore, 20(S)-PPD activates the unfolded protein response and triggers both intrinsic and extrinsic apoptotic mechanisms in liver cancer [ 26 ]. Here, we demonstrated that 20(S)-PPD exhibits anti-tumor activity in AML cells by inducing apoptosis and inhibiting proliferation. Mechanistically, 20(S)-PPD inhibits PI3K/AKT/mTOR pathway in AML, a mechanism consistent with its effects in other cancer types such as lung, liver, and breast cancer. However, 20(S)-PPD increases p-ERK1/2 in AML. One possible explanation is that PI3K/AKT/mTOR and Ras/MEK/ERK pathway exert reciprocal negative regulation on each another. Thus, inhibiting the AKT pathway may lead to the activation of the ERK pathway. Studies indicate that inhibiting PI3K can result in elevated p-ERK1/2 levels across various cancer models [ 22 , 27 , 28 ]. Considering the function of the ERK pathway in promoting tumor cell survival [ 29 ], we hypothesized that increased p-ERK1/2 protein levels could be a potential resistance mechanism against the antitumor activity of 20(S)-PPD. Our findings suggest that 20(S)-PPD significantly impacts c-Myc expression in AML cells, likely through the inhibition of the PI3K/AKT/mTOR pathway. This pathway is frequently altered in various cancers and is known to promote protein synthesis via mTORC1, which releases the inhibitory effect of 4EBP1(eIF4E binding protein 1) on eIF4E, thereby facilitating the translation of mRNAs with complex 5'-UTRs, such as c-Myc [ 30 ]. Additionally, our results showed that 20(S)-PPD activates PERK/ATF4/CHOP pathway, a key component of the unfolded protein response (UPR) during ER stress. During ER stress, PERK is activated and phosphorylates eIF2α, inhibiting global protein translation, including that of c-Myc. This translation inhibition is a protective mechanism to relieve ER stress by reducing new protein synthesis [ 21 , 31 ]. This dual action of 20(S)-PPD, suppressing the PI3K/AKT/mTOR pathway while activating the UPR, highlights its potential as a therapeutic agent targeting multiple signaling pathways in AML. The increased levels of anti-apoptotic proteins including Bcl-2 are associated with survival and chemotherapy resistance in AML [ 32 ]. However, our study revealed that 20(S)-PPD does not affect the levels of these anti-apoptotic proteins like Bcl-2. ABT-199, a Bcl-2 inhibitor, has shown notable anticancer activity in hematological malignancies [ 33 ]. Clinical data show that ABT-199 monotherapy elicits some therapeutic response in AML patients. However, 30–40 % of AML patients still fail to respond to ABT-199-based treatment, and most who initially respond eventually relapse [ 34 ]. Therefore, targeting both c-Myc and Bcl-2 simultaneously may enhance the sensitivity of AML patients to ABT-199 therapy. Our research showed that combining 20(S)-PPD with ABT-199 significantly enhances anti-tumor effects in AML cells. This combination significantly reduces c-Myc protein and mRNA levels, further enhances apoptosis, and may thereby sensitize AML cells to ABT-199 treatment. AML patients often experience high relapse rates due to resistance to cytarabine [ 25 ]. Research has shown that in AraC-resistant AML cells, c-Myc protein levels are significantly elevated [ 16 ]. Therefore, inhibiting c-Myc protein levels may help AML patients overcome resistance to cytarabine. Our study confirmed that the combination of 20(S)-PPD and ABT-199 elicits a synergistic anti-tumor effect in AraC-R AML cells. Thus, 20(S)-PPD emerges as a highly promising candidate for combating AML, offering an innovative approach for AML therapeutic strategies.

Acute myeloid leukemia (AML) is characterized as a malignant clonal disease of blood cell precursor cells [ 1 ]. It is the most common form of leukemia in adults, with a median age of diagnosis at 68 years [ 2 ]. The overall 5-year survival rate for AML patients is approximately 30 % [ 3 ]. The standard treatment for AML has remained largely unchanged for decades, primarily consisting of the “7 + 3” regimen-a combination of cytarabine and daunorubicin along with bone marrow transplantation [ 4 ]. Recently, the FDA has approved several new drugs for the treatment of AML, targeting Bcl-2, FLT3, IDH1/2, and Hedgehog pathways [ 5 ]. However, due to resistance and limited efficacy, patients are still highly susceptible to relapse after treatment [ 6 ], highlighting the urgent need for new therapeutic strategies for AML. Panax ginseng Meyer has been a highly valuable medicinal herb for over two thousand years. It is renowned for its anti-fatigue and mental stress-relieving effects and has been widely used in the medical field [ 6 ]. Ginsenosides, the primary bioactive compounds in Panax species, are categorized into two main types, oleanane and dammarane [ 7 ]. The dammarane type can be further divided into protopanaxadiol (PPD) and protopanaxatriol (PPT) [ 8 ]. 20(S)-PPD is a major PPD-type ginsenoside that has demonstrated promising pharmacological activities [ 9 ]. Recent studies have shown that 20(S)-PPD has therapeutic potential in anti-inflammation, cardiovascular protection, redox balance maintenance, neuroprotection, and endometriosis treatment [ 10 ]. Additionally, 20(S)-PPD has been shown to exhibit anti-cancer effects in various malignancies, including liver cancer [ 11 ], breast [ 12 ], prostate cancer [ 13 ], lung cancer [ 14 ], and colon cancer [ 15 ]. However, its role in AML remains be elucidated. This study primarily investigated the anti-tumor effect of 20(S)-PPD in AML. We found that 20(S)-PPD can inhibit AML cell proliferation and induce apoptosis through multiple pathways. Additionally, it exhibits a significant synergistic effect when combined with ABT-199, both in AML cells and AraC-resistant AML cells.

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The data will be available on request.