Monoclonal antibodies against Bcl-2 (#4223), Mcl-1 (#94296), Bim (#2819), Bax (#5023), caspase3 (#14220), PARP (#9532), $\gamma$-H2AX (#9718), AKT (#9272), phospho-AKT (#13038), p21 (#2947), c-Myc (#18583), CDK2 (#2546), phospho-CDK2 (#2561), and $\beta$-actin (#4970) were purchased from Cell Signaling Technology (Danvers, MA, USA). HRP-labeled goat anti-rabbit secondary antibody (ZB-5301) was purchased from Zhongshan Golden Bridge Company (Beijing, China). Venetoclax was purchased from Selleck (Houston, TX, USA) and dissolved in Dimethylsulfoxide (DMSO) at a final concentration of 10 mM. Oridonin was purchased from MedChemExpress (Monmouth Junction, NJ, USA) and also dissolved in DMSO at a final concentration of 10 mM.

AML cell lines used in the study were as follows: The THP-1 cell line was derived from human acute monocytic leukemia cells; OCI-AML3 cells were derived from human acute myeloid leukemia cells with DNMT3A and NPM1 mutations; U-937 is a cell line exhibiting monocyte morphology that was derived from histiocytic lymphoma; MOLM13 cells were derived from human acute monocytic leukemia cells with a Flt3-ITD mutation.

Cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and authenticated by short tandem repeat (STR) profiling and cytochrome c oxidase gene analysis. All cell lines underwent mycoplasma testing. OCI-AML3, MV4-11, and MOLM13 cells were cultured in complete growth medium consisting of RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. THP-1 cells were grown in RPMI-1640 without calcium nitrate, and with 0.05 mM $\beta$-mercaptoethanol, 10% fetal bovine serum, and 1% penicillin-streptomycin.

Normal human hematopoietic stem cells were obtained from peripheral blood collected from healthy donors after mobilization with 5 µg/kg granulocyte colony-stimulating factor (G-CSF). CD34${}^{+}$ cells, representing hematopoietic stem cells, were obtained by magnetic bead sorting.

Cells were seeded in 96-well plates (5 $\times$ 10${}^3$–1 $\times$ 10${}^4$ cells/well) and treated with indicated concentrations of venetoclax or oridonin for 12, 24, or 48 h. After incubation, 10 µL of CCK-8 reagent (#KGA317-1, KegGenBioTECH, Nanjing, China) was added to each well and incubated for an additional 2–4 h. Absorbance at 450 nm was measured using a microplate reader, and the proliferation inhibition rate of venetoclax or oridonin on indicated cell lines at different time points (12, 24, and 48 h) was calculated. The concentration range of venetoclax was 2.5 nM to 160 nM for MOLM13 cells and 0.25 µM to 32 µM for the remaining cell lines. The concentration range of oridonin was 0.25 µM to 32 µM for all cell lines. This experiment was independently repeated three times. The cell proliferation inhibition rate percentage was calculated as follows: (1 – (A_experiment – A_blank)/(A_control – A_blank)) $\times$ 100%.

10 mL of mobilized human peripheral blood was decanted into a 50 mL sterile centrifuge tube. CD34${}^{+}$ hematopoietic stem cells from healthy individuals were obtained by using a magnetic bead sorting kit (Miltenyi Biotec, Bergesch Gladbach, Germany). Log-phase MOLM13 and OCI-AML3 cells were washed twice with PBS and cell density of logarithmically growing cells were adjusted to 5 $\times$ 10${}^5$/mL, and 10 mL of cells were seeded in a 10 cm culture dish. Venetoclax, oridonin, and their combination were used to subsequently treat the cells for 24 h. After this incubation period, the cells were washed twice with PBS and collected. Total RNA was isolated suing the Trizol Reagent (#15596026, Invitrogen Life Technologies, Carlsbad, CA, USA), after which the concentration, quality and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA). Three micrograms of RNA were used as input material for the RNA sample preparations. The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina, San Diego, CA, USA) by Shanghai Personal Biotechnology Cp., Ltd. (Shanghai, China). Based on these alignment results, we used HTSeq (0.9.1) statistics to compare the Read Count values on each gene as the original expression of the gene, and then used FPKM to standardize the expression. Then difference expression of genes was analyzed by DESeq (1.30.0) with screened conditions as follows: expression difference multiple $|$log2FoldChange$|$ $>$1, significant p-value 0.05. At the same time, we used R language Pheatmap (1.0.8) software package to perform bi-directional clustering analysis of all different genes of samples. We got heatmap according to the expression level of the same gene in different samples and the expression patterns of different genes in the same sample with Euclidean method to calculate the distance and Complete Linkage method to cluster. We mapped all the genes to Terms in the Gene Ontology (GO) database and calculated the numbers of differentially enriched genes in each Term. Using topGO to perform GO enrichment analysis on the differential genes, calculate p-value by hypergeometric distribution method (the standard of significant enrichment is p-value 0.05. Differentially expressed genes were further validated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis.

Cells in logarithmic growth phase were seeded at a density of 3 $\times$ 10${}^5$/mL in 6-well plates and treated with venetoclax, oridonin, or a combination of both, an untreated (blank) control group was run in parallel. Cells were harvested after 24h and total RNA was extracted using Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA). RNA purity and concentration were measured using a NanoDrop 2000 spectrophotometer, and sample integrity was checked by electrophoresis. cDNA was synthesized using a reverse transcription kit (#RR037A, TaKaRa, Kyoto, Japan) and PCR was performed using an RT-PCR kit (#RR820A, TaKara, Kyoto, Japan) on ABI 7500 Real-Time PCR System (Thermo Scientific, Waltham, MA, USA). Thermocycling was carried out in three steps: 95 °C for 30 seconds for denaturation, cycling at 95 °C for 10 seconds and 60 °C for 30 seconds, and reactions were held 60 °C for 60 seconds following thermocycling. Relative gene expression was calculated using the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method with $\beta$-actin as the internal reference standard. The sequences of the primers used for qRT-PCR are given in Table 1.

Logarithmically growing cells were seeded at a density of 3 $\times$ 10${}^5$/mL in 6-well plates and treated with oridonin alone, venetoclax alone, or a combination of both. Blank (untreated) controls were also run in parallel. After 24 h of treatment, the cells were collected, washed twice with PBS, and lysed using pre-cooled cell lysis buffer. The lysates were centrifuged at 1400 g for 15 minutes at 4 °C, and the resulting supernatants were collected as whole cell protein lysates. Protein concentration was determined using a BCA assay kit, and equal concentrations of protein were loaded onto SDS-PAGE gels for electrophoresis. The proteins were subsequently transferred to PVDF membranes (Millipore, Billerica, MA, USA), membranes were subsequently blocked with 5% skim milk for 1 hour at 4 °C, and finally incubated with primary antibodies overnight. After washing with TBST buffer three times, the membranes were incubated with secondary antibodies for 1 hour at room temperature. The membranes were washed three times with TBST, and chemiluminescent reagents were used for detection. Protein bands were visualized using enhanced chemiluminescence (ECL) and recorded using X-ray film and an Amersham Imager 600 imaging system (GE Healthcare, Chicago, IL, USA). Grayscale values were measured using Image J software (version 1.46R, National Institutes of Health, Bethesda, MD, USA).

Female immunodeficient NTG mice (NOD/ShiLtJGpt-Prkdc${}^{\text{em26Cd52}}$Il2rg${}^{\text{em26Cd22}}$/Gpt) (5–6 weeks old) were purchased from Sibeifu Biotechnology Co., Ltd (Beijing, China). 5 $\times$ 10${}^6$ OCI-AML3 cells stably expressing luciferase were injected into the tail vein of NTG mice, and in vivo imaging was performed on the fourth day to confirm successful establishment of the AML xenografts. Following this, mice were randomly divided into four groups of nine, including a blank (untreated) control group, a venetoclax monotherapy group, an oridonin monotherapy group, and a combination therapy group. Venetoclax was administered orally at a dose of 100 mg/kg body weight once daily, and oridonin was administered orally at a dose of 40 mg/kg body weight once daily. In the combination group, the doses of venetoclax and oridonin were the same as those used in the monotherapy groups. In vivo imaging was performed twice a week to observe tumor burden and fluorescence intensity in the affected organs before and after drug treatment, and the mouse weight and survival were recorded. On the 11th day, three mice from each group were sacrificed and mononuclear cells were obtained by grinding the affected spleen. Bone marrow cells were obtained by flushing mouse femurs. CD33(#366608)/CD45(#304026) antibodies (Biolegend, San Diego, CA, USA) were subsequently used to label the cells, and flow cytometry was used to analyze the leukemia cell proportion in the bone marrow and spleen of the mice in each group. Remaining mice were monitored for weight and survival, and survival curves were plotted. These animal experiments were conducted in accordance with the project permit (No. 20200322) granted by the Zhengzhou University Committee on Life Science Ethics, and complied with the Zhengzhou University guidelines for animal care and use.

To investigate the effects of venetoclax and oridonin on AML cell proliferation, we initially used the CCK-8 assay to quantify the effects of different concentrations of venetoclax (Fig. 1A,C) and oridonin (Fig. 1B,D) on the proliferation of MOLM13, OCI-AML3, U937, and THP-1 cell lines at 12 h, 24 h, and 48 h pot-treatment. Results obtained indicate that venetoclax or oridonin alone can inhibit the viability and proliferation of AML cells, exhibiting both time and concentration-dependent effects. The half-maximal inhibitory concentrations (IC50) values were calculated using Graphpad Prism 6 software (GraphPad Software, San Diego, CA, USA) (Fig. 1E) and showed that in these four cell lines, the IC50 value of Venetoclax for the MOLM13 cells at 24 h was 4.7 nM, indicating that MOLM13 cells were significantly sensitive to venetoclax. In contrast, the measured IC50 values of venetoclax for the OCI-AML3, U937, and THP-1 cells at 24 h were 3.9 µM, 5.0 µM, and 4.3 µM, respectively. These findings indicate resistance to venetoclax in these 3 AML cell lines. The IC50 values (95% confidence interval) of oridonin for the four cell lines showed relatively small changes and ranged from 0.5 µM to 5 µM.

Fig. 1.

The inhibitory effects of venetoclax and oridonin on acute myeloid leukemia (AML) cell line proliferation using a CCK8 assay. MOLM13, OCI-AML3, U937, THP-1 cells were treated with venetoclax (concentrations ranging from 0.5 nM to 64 µM) or oridonin (concentrations ranging from 0.125 µM to 64 µM). (A) Cell viability of AML cells after treatment with different concentrations of venetoclax for 12 h, 24 h, and 48 h. (B) Cell viability of AML cells after treatment with different concentrations of oridonin for 12 h, 24 h, and 48 h. (C) Proliferation inhibitory rate of AML cells after treatment with different concentrations of venetoclax. (D) Proliferation inhibitory rate of AML cells after treatment with different concentrations of oridonin. (E) IC50 (95% confidence interval) values of venetoclax and oridonin on AML cells after 12 h, 24 h, and 48 h of treatment. Each experiment was repeated independently three times. Con, control; Ven, venetoclax; Orid, oridonin; Comb, combination of Ven and Orid.

Above results indicate that both venetoclax and oridonin can inhibit the growth of AML cells. To investigate whether the combination of these two drugs has a synergistic effect on promoting AML cell apoptosis, we used an Annexin V-FITC and PI double staining assay to measure apoptosis when AML cells were treated with the two drugs alone or in combination. The results indicated that both venetoclax (Fig. 2A) and oridonin (Fig. 2B) alone induce AML cell apoptosis. After treatment with a combination of the two drugs, the induction of cell apoptosis was further enhanced. Specifically, the combination index (CI) values, calculated using Compusyn software, indicated a CI of less than 1, indicating a synergistic effect on promoting apoptosis (Fig. 2C). The CI theorem of Chou-Talalay offers a quantitative definition for additive effect (CI = 1), synergism (CI 1), and antagonism (CI $>$1) in drug combinations [11].

Fig. 2.

Annexin V-FITC and Propidium Iodide (PI) double staining assay was used to quantify apoptosis in AML cells treated with different concentrations of venetoclax and oridonin. (A) Apoptosis rate of AML cells treated with venetoclax (0~32 µM) for 24 h. (B) Apoptosis rate of AML cells treated with oridonin (0 µM~32 µM) for 24 h. (C) Changes in AML cell apoptosis following treatment with different concentrations of venetoclax and oridonin for 24 h, and the combination index (CI) was calculated. * indicates CI 1. (D) Changes in mRNA levels of indicated Bcl-2 family members in AML cells treated with venetoclax and oridonin for 24 h (Mean $\pm$ SD; single */#: p 0.05; double **/##: p 0.01; */**: Ven/Orid/Comb vs Con; #/##: Comb vs Ven/Orid). (E,F) MOLM13 cells were treated with 2.5 nM venetoclax and OCI-AML3 cells were treated with 4 µM venetoclax, while MOLM13 cells were treated with 2 µM oridonin and OCI-AML3 cells were treated with 4 µM oridonin. Subsequently, apoptosis-related proteins and Bcl-2 family members were detected by western blotting 24 h after treatment. Con, control; Ven, venetoclax; Orid, oridonin; Comb, combination of Ven and Orid.

Based on the synergistic induction of cell apoptosis by the two drugs, we selected 2.5 nM venetoclax and/or 2 µM oridonin for single or combined treatment of MOLM13 cells, and 4 µM venetoclax and/or 4 µM oridonin for single or combined treatment of OCI-AML3 cells. Further, we used RT-qPCR to detect changes in expression of Bcl-2 family-related genes in drug-treated cells. The results showed that there was no significant effect of venetoclax on the transcription levels of Bcl-2 and Mcl-1 in MOLM13 cells, but that venetoclax induced an increase in Mcl-1 transcript abundance in OCI-AML3 cells. However, when combined with oridonin, the transcription level of Mcl-1 decreased significantly in OCI-AML3 cells. The gene expression levels were represented as Mean $\pm$ SD. The statistical analysis was conducted using t-test to assess the significance of the expression differences (Fig. 2D).

Western blotting was also used to detect changes in expression of apoptosis-related proteins and Bcl-2 family regulatory proteins following drug treatment. Results obtained indicate that venetoclax alone increased the levels of various markers of apoptosis such as cleaved (cf) Caspase3, cf-PARP, and $\gamma$-H2AX proteins in MOLM13 and OCI-AML3 cells. Of note, a more significant effect of venetoclax on MOLM13 was noted, and oridonin further enhanced this effect. This indicates that venetoclax can cause DNA damage, induce cell apoptosis, and this effect is further enhanced when combined with oridonin (Fig. 2E).

The expression levels of Bcl-2 family proteins were examined using western blotting. Results obtained are shown in Fig. 2F. In regards to Mcl-1, treatment with venetoclax alone increased the expression of Mcl-1, with a lower increase in sensitive MOLM13 cells compared to more resistant OCI-AML3 cells. Various Mcl-1 phosphorylation sites can alter the funcrion of this protein. For example, Mcl-1 phosphorylation at Ser159 (Mcl-1s) makes the Mcl-1 protein unstable and easily degraded, while phosphorylation at Thr163 (Mcl-1t) increases Mcl-1 stability and promotes an anti-apoptotic role [12]. In this study, after treatment with venetoclax alone, the abundance of Mcl-1t decreased in MOLM13 cells, while the abundance of Mcl-1s increased. In OCI-AML3 cells, Mcl-1s did not exhibit a significant change, but Mcl-1t was observed to be increased. When venetoclax was combined with oridonin, both cell lines showed an increase in Mcl-1s and a decrease in Mcl-1t abundance. We also observed that after 24 h of treatment with venetoclax, the abundance of the anti-apoptotic protein Bcl-xl decreased, and this effect was further enhanced when combined with oridonin. When we studied the pro-apoptotic protein Bax we noted that treatment with venetoclax or oridonin alone increases Bax abundance, and Bax abundance levels further increased when these two drugs were combined. Similarly, the pro-apoptotic protein Bak showed that with oridonin alone an increase in Bak abundance was noted. Taken together, these findings indicate that oridonin can synergize with venetoclax to promote AML cell apoptosis by, in part, downregulating anti-apoptotic proteins.

Mitochondria play an important role in the activation of apoptosis. In the early stages of apoptosis, the permeability of the mitochondrial membrane changes and the opening of permeability transition pores cause a decrease in the mitochondrial transmembrane potential. In normal cells, the mitochondrial membrane potential is maintained at a relatively high level, resulting in the accumulation of the dye JC-1 in the mitochondrial matrix and the formation of JC-1 aggregates that produces red fluorescence that can be detected by flow cytometry. During early apoptosis, the mitochondrial membrane potential decreases, preventing the accumulation and aggregation of JC-1 in the mitochondrial matrix. As a result, JC-1 exists as monomers, leading to the production of green fluorescence [13]. We sought to detected the effects of venetoclax and oridonin on the mitochondrial membrane potential of AML cells. The results (Fig. 3A,B) showed that when venetoclax was combined with oridonin, there was a significant decrease in mitochondrial membrane potential in the MOLM13, OCI-AML3, U937, and THP-1 cell lines, and the differences were statistically significant (p 0.05). The values were represented as Mean $\pm$ SD. The statistical analysis was conducted using t-test.

Fig. 3.

Changes in mitochondrial membrane potential of AML cells treated with venetoclax or oridonin alone, or in combination. (A) Venetoclax and Oridonin were applied to four cell lines, including MOLM13, OCI-AML3, U937, and THP-1 for 24 h (the concentration of venetoclax was 2.5 nM, 4 µM, 8 µM, 1 µM in MOLM13, OCI-AML3, U937, and THP-1 cells, respectively; the concentration of oridonin was 2 µM, 4 µM, 8 µM, 4 µM, respectively), and the ratio of JC-1 monomers/polymers was detected by flow cytometry. (B) Statistical differences between experimental groups and control groups were compared using t-test (*: p 0.05, **: p 0.01).

We next conducted transcriptome sequencing to explore the molecular mechanism that governs the synergistic effect of oridonin and venetoclax in promoting apoptosis in AML cells. Previous results determined that different AML cell lines have different sensitivities to venetoclax, with MOLM13 being sensitive and OCI-AML3, U937, and THP-1 showing resistance. Our findings indicate that oridonin can synergize with venetoclax to induce apoptosis in AML cells; thus, to investigate the mechanism that controls this sensitivity to venetoclax, we selected venetoclax-sensitive MOLM13 cells, venetoclax-resistant OCI-AML3 cells, and normal peripheral blood CD34${}^{+}$ hematopoietic stem cells as controls. We subsequently performed transcriptomic sequencing analysis to explore this mechanism. Transcriptomic sequencing indicates that both MOLM13 and OCI-AML3 cells have many differentially expressed genes when compared to normal hematopoietic stem cells, and there are also differential gene expressions between the two AML cell lines (Fig. 4A). Through GO functional enrichment analysis, we uncovered functional differences in MAPK signaling pathway regulation, cell cycle arrest, and p53-associated apoptosis regulation between OCI-AML3 and MOLM13 cells (Fig. 4B,C).

Fig. 4.

Differential gene expression analysis and enrichment results of transcriptome sequencing. (A) Differential gene expression in OCI-AML3, MOLM13, and normal hematopoietic stem cells. (B,C) Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes between MOLM13 and OCI-AML3. (D) Differential gene expression before and after drug treatment of OCI-AML3 cells. (E) KEGG enrichment analysis of differential gene expression between combination drug treatment and control groups. (F) KEGG enrichment analysis of differential gene expression between th4 combination drug group and venetoclax monotherapy group. SC, stem cells from healthy person; Con, control; Ven, venetoclax; Orid, oridonin; Comb, combination of Ven and Orid.

To investigate which of these differences contribute to drug resistance, we treated OCI-AML3 cells with 4 µM venetoclax, 4 µM oridonin, or a combination of both, and explored their molecular mechanisms through transcriptome sequencing. The results showed that the quantitative gene expression changes induced by venetoclax were less than those induced by oridonin in OCI-AML3 cells after drug treatment (Fig. 4D). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that compared with the venetoclax single-drug group, the combination therapy group also contained differentially expressed genes in the p53 and MAPK signaling pathways as well as cell cycle regulation (Fig. 4E,F). These transcriptome sequencing results suggested that the PI3K/AKT and p53 signaling may affect the sensitivity of AML cells to venetoclax.

Based on the results of our sequencing, we used flow cytometry to detect changes in cell cycle distribution after drug treatment in MOLM13 and OCI-AML3 cells. The effects of these drugs on cell cycle dynamics were different in the various AML cell lines used. We observed that venetoclax had no significant effect on cell cycle distribution. In contrast, oridonin arrested MOLM13 cells in the G1 phase and OCI-AML3 cells in the G2 phase of the cell cycle. In addition, when the two drugs were used in combination, the cell cycle arrest effect observed with oridonin treatment alone was further enhanced in both MOLM13 cells (arrested in the G1 phase) and OCI-AML3 cells (arrested in the G2 phase) (p 0.01). Although oridonin also arrested U937 cells in S phase (p 0.01), this effect was weakened when the two drugs were used together. In THP-1 cells, oridonin arrested cells in the G2 phase, and the addition of venetoclax did not significantly alter this effect. These results indicate that the combination of venetoclax and oridonin can result in a significant arrest of the cell cycle of AML cells.

Furthermore, we used western blotting to detect changes in the expression of several cell cycle-related proteins. Namely, we examined p21, CDK2, phospho-CDK2, and c-Myc in MOLM13 and OCI-AML3 cells after drug treatment. The results showed that the expression of p21 was down-regulated after venetoclax alone, most notably in MOLM13 cells. Oridonin was found to up-regulate p21 when used alone, and the combination of the two drugs increased the expression of p21 compared to venetoclax alone. Oridonin down-regulated the expression of cyclin D1, and both drugs down-regulated the expression of c-Myc. When used together, this effect was further enhanced as the two drugs had no significant effect on the total protein expression of CDK2, but oridonin could inhibit the phosphorylation of CDK2 in MOLM13 cells. This led to a decrease in the level of phospho-CDK2 and G1 cell cycle arrest. In OCI-AML3 cells, oridonin up-regulated phosphorylation of CDK2, leading to an increase in the level of phospho-CDK2. This finding may help explain the different cell cycle arrest stages induced by venetoclax and oridonin in these two cell lines (Fig. 5C).

Fig. 5.

Cell cycle alterations in AML cells treated with venetoclax or oridonin alone, or in combination. (A) Venetoclax and oridonin were applied to MOLM13, OCI-AML3, U937, and THP-1 for 24 h. The concentration of venetoclax was 2.5 nM, 4 µM, 8 µM, 1 µM in MOLM13, OCI-AML3, U937, and THP-1 cells, respectively. The concentration of oridonin was 2 µM, 4 µM, 8 µM, 4 µM, respectively. Cell cycle dynamics were obtained by flow cytometry. (B) Statistical differences between experimental groups and control groups, as well as between combination groups and single drug groups, in each cell line were compared using t-test (* experimental group vs control group; # combination group vs single drug group; */#: p 0.05, **/##: p 0.01). (C) Expression of indicated cell cycle-associated proteins by western blotting. Con, control; Ven, venetoclax; Orid, oridonin; Comb, combination of Ven and Orid.

The results of transcriptomic sequencing suggested that the PI3K/AKT and p53 signaling pathways may affect the sensitivity of AML cells to venetoclax. To investigate the mechanism of action, we harvested MOLM13 and OCI-AML3 cells treated with venetoclax, oridonin, or combination for 24h, extracted total protein, and performed western blotting analysis. The results showed that both venetoclax and oridonin can inhibit AKT phosphorylation, decrease phospho-AKT abundance. However, we noted that this effect is enhanced in MOLM13 cells. When the two drugs were used in combination, the inhibitory effect was further increased (Fig. 6A).

Fig. 6.

Expression of key molecules in the MAPK/AKT and p53 pathways. (A) The effects of venetoclax and oridonin on indicated protein abundance in AML cells was investigated by western blotting. (B) Western blotting was used to examine AKT phosphorylation following 4 µM oridonin. Note this effect was reversed by pre-treatment with 10 µM SC-79 for 1 h. (C) Flow cytometry was used to detect that oridonin could induce apoptosis in OCI-AML3 cells, but this effect was abrogated by pre-treatment with SC-79 for 1 h (**: p 0.01).

To verify the role of the AKT signaling in this process, we added the AKT agonist SC-79 to overcome the inhibitory effect of oridonin on AKT. SC-79 is an AKT agonist that induces phosphorylation of AKT at Thr308 and Ser473 in the cytoplasm and promotes downstream signaling pathway activity. We performed this experiment using the OCI-AML3 cell line which is resistant to venetoclax, and treated cells with 4 µM oridonin alone, 10 µM SC-79 alone, or the combination of the two by pre-treating cells with SC-79 for 1 h before adding oridonin. The level of AKT phosphorylation and cell apoptosis rate were measured 24h later. The results showed that SC-79 alone could increase the abundance of phospho-AKT, and when used in combination with oridonin, it could reverse the inhibitory effect of oridonin on AKT phosphorylation (Fig. 6B). Co-culturing SC-79 with oridonin reduced the apoptotic response induced by oridonin (Fig. 6C).

The signaling pathways in AML cells influenced by oridonin and venetoclax are shown in Fig. 7.

Fig. 7.

The signaling pathways impacted by combination treatment with venetoclax and oridonin in AML cells. Orid, oridonin; Ven, venetoclax.

A total of 5 $\times$ 10${}^6$ OCI-AML3 cells stably expressing luciferase were injected into immunocompromised NTG mice via tail vein injection. On the fourth day, in vivo imaging was performed to confirm successful cancer cell engraftment. Subsequently, mice were subjected to drug treatment, and biweekly in vivo imaging was conducted to quantify fluorescence intensity. The results indicated that there was no statistically significant difference in fluorescence intensity between the animal groups treated with single-agent venetoclax or oridonin when compared to the control group (p $>$ 0.05); however, the difference between the combination therapy group and the control group was statistically significant (p 0.01) (Fig. 8A,B). This finding suggests that venetoclax and oridonin combination therapy could effectively reduce tumor burden in AML xenografted mice. On the eleventh day of treatment, three mice from each group were euthanized, and the size of the spleen was measured. We observed that the spleens of mice in the treatment groups showed enlargement due to infiltration of AML cells. Nevertheless, after receiving the combination drug treatment, a noticeable reduction in tumor burden was observed, accompanied by a shrinkage in spleen size (Fig. 8C). To assess the changes in the proportion of myeloid progenitor cells, bone marrow cells, and spleen grinding cells, cells were labeled with CD33/CD45 antibodies and subsequently incubated. The flow cytometry analysis revealed an increase in the proportion of CD33/CD45 double-positive cells, indicating myeloid progenitor cells was higher in the spleen compared to the bone marrow. In the femoral bone marrow, venetoclax monotherapy group exhibited a significant decrease in the proportion of CD33/CD45 double-positive cells compared to controls (p 0.05). However, there was no statistically significant difference observed between the oridonin monotherapy group and the control group. The combination therapy group demonstrated a further decrease in CD33/CD45 double-positive cells was noted compared to the control group and this was a significant difference (p 0.01). In the spleen, no statistically significant difference was observed in the proportion of CD33/CD45 double-positive cells between either of the monotherapy groups and the control group (p $>$ 0.05). However, after receiving combination therapy, a statistically significant decrease in the proportion of CD33/CD45 double-positive cells was observed when compared to either the control group or the monotherapy groups (p 0.05) (Fig. 8D,E). The lungs, liver, and kidneys of the mice were dissected and immunohistochemistry staining was performed using a human-specific (hCD45, #CY5311, Abways, Shanghai, China) antibody. The expression of hCD45 in the combination therapy group was lower than that in the control group (Fig. 8G). Survival time of the mice was recorded, and the results demonstrated a significant extension in the survival time of the combination therapy group compared to controls (p 0.01). However, there was no statistically significant differences observed in animal survival time between the combination and the monotherapy groups (p $>$ 0.05) (Fig. 8F).

Fig. 8.

The effect of venetoclax and oridonin on AML xenografts in mice. (A) In vivo imaging of AML-implanted mice treated with venetoclax and oridonin alone, or in combination. (B) Fluorescence intensity values measured in AML-implanted mice. (C) Spleens were obtained after euthanasia on day 11. (D) Flow cytometry scatter plot of CD33${}^{+}$CD45${}^{+}$ cells in bone marrow and spleen on day 11. (E) Proportion of CD33${}^{+}$CD45${}^{+}$ cells in bone marrow and spleen on day 11 (Mean $\pm$ SD, single */#: p 0.05; double **/##: p 0.01; */**: Ven/Orid/Comb vs Con; #/##: Comb vs Ven/Orid). (F) Kaplan-Meier survival curves of AML xenografted mice treated with venetoclax, oridonin, and their combination. Results were analyzed by Graphpad Prism software. (G) hCD45 staining in lungs, elbows, and kidneys of mice on day 11. Con, control; Ven, venetoclax; Orid, oridonin; Comb, combination of Ven and Orid.