The anti-leukemic effects of vitamin K4 by modulating the JAK/STAT signaling pathway

Abstract

Background

Vitamin K4 (VK4) is a hemostasis medicine used in clinical practice. It has also been reported to possess anti-cancer properties in types of solid tumors, however, its antitumor effects in leukemia cells are unknown. We aimed to investigate the anti-leukemia role of VK4 and the involved mechanisms.

Methods

CCK-8 was used to detect the cytotoxic activity, and flow cytometry was applied to test cell cycle, apoptosis and mitochondrial membrane potential (MMP) level in human leukemia cells. Western bolting was conducted to examine the protein expression level. A xenografting leukemia model was established to explore the anti-leukemia effects triggered by VK4 in vivo.

Results

VK4 inhibited the proliferation of human leukemia cell lines by inducing the cell cycle arrest and mitochondrial-mediated apoptosis in HL60, Jurkat and K562 cells. MMP level was also observed to be reduced under the treatment of VK4 in these three leukemia cell lines. Further studies found that VK4 inhibited the activation of JAK-STAT signaling pathway by decreasing phosphorated JAK2 and STAT3/5. The pretreatment of AG490, an inhibitor for JAK2/3 and STAT3 could significantly reverse the effect of VK4 on cell proliferation in HL60 and K562 cells. Moreover, data from HL60-xenograft model in BALB/c nude mice demonstrated that VK4 could inhibit the proliferation of HL60 in vivo in a dose-dependent manner.

Conclusion

VK4 exhibited a significant anti-leukemia activity by inducing cell cycle arrest and apoptosis in human leukemia cell lines through inhibiting the JAK/STAT signaling pathway. The anti-leukemia effects both in vitro and in vivo indicated that VK4 might be developed as a promising therapeutic candidate against leukemia.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-026-04356-6.

Introduction

Leukemia is a malignant disease of the blood and bone marrow, which is characterized by the distorted proliferation and accumulation of clonal leukemia cells in the bone marrow and other hematopoietic tissues [1]. Although the treatments including chemotherapy, immunotherapy, and/or targeted therapy for leukemia greatly increase the number of complete remissions and long-term disease-free survivors, it is still a severe disease that significantly threats global public health [2]. Data from the latest global cancer statistics in 2025 indicates that leukemia ranks 10th in incidence and 7th in mortality, with 66, 890 estimated new cases and 23, 540 estimated deaths in the United States [3]. It is urgent to develop novel drugs against hematologic oncology.

The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) is one of the most conserved signaling pathway that plays significant roles in biological processes, including cell division, differentiation, and apoptosis [4]. Dysregulation of the JAK-STAT signaling pathway with hyperactivation usually leads to the development and progression of multiple cancers [5]. It is demonstrated that the JAK2-STAT3/5 signaling pathway play a critical role in promoting the advancement of leukemia [6], and targeting therapies against JAKs or STATs are widely studied in leukemia diseases [7].

Vitamin K (VK) is a group of compounds with similar structures that feature a 2-methyl-1,4 naphthoquinone ring along with an aliphatic chain that varies in nature, including VK1, VK2, VK3, and VK4, among which, VK1 and VK2 are natural products [8–10], while VK3 and VK4 are synthetic derivatives from the chemical backbone of VK2 [11]. The common physiological functions of VKs include promoting blood coagulation and participating in bone metabolism [12, 13]. Recently, an increasing number of studies have revealed their antitumor effects. It was suggested that VK1, VK2, and VK3 can inhibit the proliferation and growth of types of cancers through inducing cellular apoptosis [14–19]. In addition, VKs were also reported to exhibit significant anti-leukemia effects. Specifically, VK2 can induce apoptosis in NB4 cells (a type of human AML cell line) and enhance the anti-leukemic activity of all-trans retinoic acid (ATRA) [20]. Combination therapy with VK2 and ATRA has been reported to achieve complete remission in patients with relapsed acute promyelocytic leukemia [21]. Additionally, it was also reported that VK2 could trigger both apoptosis and autophagy in HL60 cells to inhibit cell proliferation [22], while VK3 exerted anti-leukemic effects through selectively targeting of mitochondrial complexes [23]. The cytotoxic effects of VK2 and VK3 against leukemia cells are associated with apoptosis induction and cell cycle arrest [24].

VK4, also named Menadiol Diacetate, is a water-soluble synthetic derivative of VK2 and was initially utilized as a clinical hemostatic agent [11]. Compared with other members in the VK family, the antitumor roles of VK4 are rarely reported, and only types of solid tumors are found to be affected by VK4. In 2013, Jiang et al. first demonstrated VK4’s anticancer activity in PC-3 cells derived from prostate cancer [25]. In 2017, Di et al. found that VK4 could inhibit the proliferation of U2OS osteosarcoma cells and induce apoptosis through mitochondrial dysfunction [26]. However, to date, no studies has been focused on its anti-leukemia effects. In this study, we first investigated the anti-cancer effects of VK4 on leukemia cells in vitro and in mice xenograft model in vivo. The underlying molecular mechanisms suggested that VK4 could block the JAK-STAT signaling pathway to induce the mitochondria-mediated apoptosis, leading to the suppression of cell proliferation in myeloid leukemia HL60 and K562 cells. These findings indicated that VK4 could be a promising candidate targeting the JAK-STAT signaling pathway, which was deserved to further exploration in the therapy of leukemia.

Materials and methods

Reagents

VK4 and AG490 (inhibitor for JAK2/3 and STAT3) were purchased from Med. Chem. Express; fetal bovine serum and cell culture medium (RPMI 1640) were purchased from Gibco; the Cell Counting Kit-8 (CCK-8) Assay Kit was obtained from Bogreen Biotechnology Co (Dalian, China); propidium iodide (PI) for cell cycle detection was acquired from Sigma-Aldrich; the Annexin V-Alexa Fluor 488/PI apoptosis assay kit was bought from 4 A Biotech; the Mitochondrial Membrane Potential Detection Kit was attained from BestBio. The primary antibodies against human GAPDH (Cat#: 60004-1-Ig), CDK1 (Cat#: 10762-1-AP), Caspase 3/ Cleaved-Caspase 3 (Cat#: 19677-1-AP), Caspase 8 (Cat#: 13423-1-AP), Caspase 9/Cleaved-Caspase 9 (Cat#: 10380-1-AP), BAX (Cat#: 50599-2-Ig), BAD (Cat#: 10435-1-AP) and Bcl-2 (Cat#: 26593-1-AP) were obtained from Proteintech, China; the primary antibodies against human CDK4 (Cat#: 12790), Cyclin A2 (Cat#: 91500), Cyclin B1 (Cat#: 12231), Cyclin E2 (Cat#: 4132), PARP (Cat#: 9542), XIAP (Cat#: 14334), Survivin (Cat#: 2808), MCL-1 (Cat#: 5453) and Bcl-xL (Cat#: 2764) were purchased from Cell Signaling Technology; the primary antibodies against human CDK2 (Cat#: ab32147) was purchased from Abcam; the primary antibodies against JAK2 (Cat#: T55287), phospho-JAK2 (Cat#: TU390534), STAT3 (Cat#: T55292), phospho-STAT3 (Cat#: T56566), STAT5A/B (Cat#: T55874) and phospho-STAT5 (Cat#: T55569) were purchased from Abmart, China; the primary antibodies against human CD33 (Cat#: abs172266) was purchased from Absin, China.

Cell culture

Human acute myeloid leukemia (AML) cell line HL60, human acute lymphocytic leukemia (ALL) cell line Jurkat, and human chronic myeloid leukemia (CML) cell line K562 were sourced from the cell bank of the Typical Culture Preservation Committee of the Chinese Academy of Sciences. All cell lines were cultured in complete medium (RPMI-1640 medium that was supplemented with 10% fetal bovine serum) in an incubator at 37 °C with 5% CO₂.

Isolation of primary peripheral blood mononuclear cells and leukemia cells

Normal peripheral blood mononuclear cells (PBMCs) are separated from the peripheral blood samples of healthy volunteers and primary leukemia cells are isolated from the bone marrow blood samples of clinical leukemia patients [27, 28]. The research protocol had been reviewed and approved by the Research Ethics Committee of the First Affiliated Hospital of Gannan Medical University, with Ethics No. LLSC-2024 No. 283. Briefly, mononuclear cells from BM were separated by density-gradient centrifugation with Ficoll-Hypaque (Cytiva, Uppsala, Sweden), and cultured in IMDM (HyClone, Logan, Utah, USA) supplemented with 15% FBS (Gibco, Australia), 4 mM L-Glutamine (Gibco, Brazil), 100 ng/mL human IL-3 (Proteintech, China), 10 ng/mL human IL-6 (Proteintech, China), 50 ng/mL human M-CSF (Proteintech, China), TPO (Proteintech, China), 50 ng/mL human FLT3-ligand (Proteintech, China), 50 ng/mL human SCF (Proteintech, China) and 1% penicillin and streptomycin. PBMCs were cultured in PRIM-1640 supplemented with 10% FBS and 1% penicillin and streptomycin.

Cell viability assay

The CCK-8 Enhanced Assay Kit was employed to evaluate the cytotoxic effect of VK4 in leukemia cell lines, PBMCs and primary leukemia cells. Cells in the logarithmic growth stage were seeded into 96-well plates and treated with VK4 at the indicated concentrations for 24 h. Then the cell viability was detected according to the manufacturer’s instructions using a Multifunctional Microplate Reader (Thermo Fisher Scientific). The data was analyzed using GraphPad Prism 10.0 and the half-maximal inhibitory concentration (IC₅₀) was calculated according to the relative survival curve.

Cell cycle assay

Leukemia cells in the logarithmic growth stage were seeded in 6-well plates and treated with the indicated concentrations of VK4 or vehicle (< 0.1% DMSO) for 12 h. And then cells were collected and washed by cooled PBS, with 70% ethanol fixed at -20 °C overnight. Cells were centrifuged at 4 °C and resuspended in PBS with RNase A at 37 °C for 30 min. Subsequently, cells were incubated with Propidium Iodide (PI) solution (10 mg/ml) in dark at room temperature. After a filtration through a 300-mesh nylon membrane, cells were measured by FACS Calibur flow cytometer (BD Biosciences). Data was analyzed by FlowJo_v10.9.0 analysis software.

Apoptosis detection

Leukemia cells were plated in 6-well plates and pretreated with AG490 or not for 12 h, and then treated with the indicated concentrations of VK4 or vehicle (< 0.1% DMSO) for another 24 h. Cells were washed by cooled PBS and resuspended in Binding buffer to adjust cell density. Then Annexin V and PI were added to the samples in the flow tubes. The apoptosis cell numbers were measured by FACS Calibur flow cytometer (BD Biosciences). Data was analyzed by FlowJo_v10.9.0 analysis software.

Mitochondrial membrane potential level detection

Intracellular mitochondrial membrane potential (MMP) levels were measured using the MitoProbe™ JC-1 Assay Kit for Flow Cytometry (Cat#: M34152, Invitrogen) according to the manufacturer’s instructions and previous reports [29, 30]. Briefly, Jurkat, HL60 and K562 cells were seeded in 6-well plates and treated with VK4 at the indicated concentrations for 24 h. And then cells were washed by PBS and resuspended in JC-1 staining solution at 37 °C for 30 min. Nex, cells were washed with PBS and resuspended in 500 µL PBS. Flow cytometry (FACS Calibur flow cytometer, BD Biosciences) was used to detect the intracellular MMP level. Generally, a decrease in the red/green fluorescence intensity ratio indicated the mitochondrial depolarization occurring in apoptosis. Data was analyzed by FlowJo_v10.9.0 analysis software.

Western blotting

HL60, Jurkat, K562 cells and primary leukemia cells were treated with VK4 in the indicated concentration for 24–48 h, and then collected and lysed on ice with RIPA buffer supplemented with Protease Inhibitors, phosphatase inhibitors (Cat#: 4693116001, Roche) and Phenylmethanesulfonyl fluoride (Cat#: HY-B0496, MEK). After a quantification for protein concentration using BCA assay kit (Cat#: 23227, Thermo Fisher), the lysates in sample buffer were heated at 100 °C for 5 min to make proteins denatured. An amount of 20 µg total protein per well from each sample was loaded onto 8 ~ 15% gradient SDS-PAGE gel and run for 1 ~ 2 h at 120 V until the dye front reaches the agarose layer of the gel. PVDF membrane was pre-activated with Methanol for 30 s, and then rinsed with transfer buffer before preparing the stack: lay a filter paper stack at the bottom, followed by the gel, and then the PVDF membrane, at last another stack of filter paper on the top. A wet transfer was performed in cold transfer buffer running at 0.2 ~ 0.3 Amps for 60 min. Afterwards, the PVDF membrane was blocked with 5% skimmed milk in PBS containing 0.1% Tween-20 (PBST) for 2 h at room temperature, and then incubated with specific primary antibodies at 4 °C overnight, following by an incubation of secondary antibodies for 2 h at room temperature. At last, the membrane was submerged in chemiluminescence (ECL) substrate and the blots were observed using a Molecular Imager ChemiDocTM XRS imaging System (Bio-Rad).

Bio-Layer Interferometry (BLI) to test the interaction between VK4 and JAK2

Human recombinant 6×His-JAK2 protein (752–1132 aa) was purchased from Targetmol (Cat#: TMPH-02274), and dissolved in PBS to make a concentration of 100 µg/mL. After pre-wetting the Ni-NTA (Nitrilotriacetic acid) biosensor with PBST to record the baseline, the JAK2 in 96-well black F-bottom plates were directly immobilized on the Ni-NTA biosensor. VK4 was diluted by PBST (containing 5% DMSO and 0.02% Tween 20) to 0.1, 0.3, 1.0, 3.0, 10.0 and 30.0 µM with the final volume of 200 µ L per well. At the same time, an equal volume of PBST (containing 5% DMSO and 0.02% Tween 20) was added to wells and set as the control group. The baseline, as sociation and dissociation steps were operated for 90 s respectively [31]. The data were acquired and analyzed by ForteBio Octet Data Acquisition and Data Analysis software (version 11.0).

Animal model

Three-week old female BALB/c nude mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Science and Technology Co. (CNAS Certificate No. LA0021, Jiangsu, China) and fed in a specific pathogen-free (SPF) environment and treated in accordance with the guidelines of Animal Ethics Committee of First Affiliated Hospital of Gannan Medical University (Ethics No. LLSC-2024 No. 078). After an acclimatization in local animal experimentation center for one week, the mice were pretreated with100 mg/kg cyclophosphamide for two successive days. After that, a volume of 100 µL HL60 cells resuspend in PBS were injected into the mice tail vein (5 × 10⁶ cells per mouse) to establish xenograft model [32]. Venous blood was collected from the tail every 7 days. Peripheral blood smears were prepared from Venous blood every 7 days to monitor the leukemia cells colonization in vivo. Approximately 2 weeks post HL60 vaccination, mice were randomly assigned to vehicle control group (10% dimethyl sulfoxide (DMSO), 40% polyethylene glycol 300 (PEG 300), 5% Tween-80, and 45% saline (0.9% NaCl), n = 8) and VK4 treatment group (2.5, 5.0, or 10.0 mg/kg, n = 8 ~ 10/group). VK4 or vehicle were admitted by intraperitoneal injection daily at the indicated dosage. Peripheral blood smears were examined once a week to monitor the leukemia proliferation in vivo. At the end of treatment (28 days), mice were euthanized by cervical dislocation following anesthesia (intraperitoneal injection of sodium pentobarbital, 80 mg/kg). The liver, spleen, and kidney tissues were harvested to stain with hematoxylin-eosin (HE), and the spleen was subjected to immunohistochemical experiments to detect the expression level of CD33.

Mice survival analysis

The HL60 xenograft model was established as described above (see Animal model for details). Subsequently, the tumor-bearing mice were randomly divided into four groups (n = 10 per group): a vehicle control group, and groups treated with VK4 at doses of 2.5, 5, or 10 mg/kg. For survival analysis, mice were monitored daily for clinical signs of leukemia progression, with the first day of VK4 administration defined as the starting point and the date of euthanasia (humane endpoints) considered as the event date for generating Kaplan-Meier survival curves. According to the previous studies for leukemia mouse model [33, 34] and NIH Guidelines for Endpoints in Animal Study Proposals (2019), humane endpoints were defined as the presence of any of the following criteria:

1. Body condition score (BCS) ≤ 2 on a 1–5 scale (pronounced vertebral ridge and palpable pelvic bones) [35].

2. Body weight loss exceeding 20% of initial body weight.

3. Severe behavioral changes including hunched posture persisting for > 24 h with markedly decreased activity (failure to move when stimulated), lateral recumbency (inability to right themselves within 30 s), or unstable gait.

4. Clinical signs including labored breathing, pale footpads (indicating severe anemia), palpable splenomegaly, or hypothermia (< 33 °C by rectal probe).

5. Complete anorexia for > 24 h.

Mice meeting any of these criteria were immediately euthanized by cervical dislocation under isoflurane anesthesia to confirm death. No animals were found dead in their cages. Mice that survived to the end of the observation period (day 76 after VK4 treatment) were censored.

Blood smear test and Wright-Giemsa staining

After HL60 cells inoculating into BALB/c nude mice, peripheral venous blood from the tail vein was collected to perform the blood smear test. Briefly, single-nucleus cells in blood were stained by Wright-Giemsa according to the to the manufacturer’s instructions (Cat#: BA4017, BaSo), and observed via a Zeiss microscope.

Hematoxylin-eosin (HE) staining

Liver and kidney Sect.  (5 μm) from BALB/c nude mice were first stained with hematoxylin for 3 ~ 5 min, subsequently stained with eosin for 15 ~ 30 s, and then observed under a Nikon ECLIPSE Ti microscope.

Immunohistochemistry Staining

Spleen Sect.  (4 μm) from BALB/c nude mice were used to determine the infiltration of leukemia cells using rabbit anti-CD33 human recombinant monoclonal antibody. Stained sections were scanned with a Zeiss microscope.

Statistical analysis

Data are presented as the mean ± standard deviation (SD) from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 10.0 (GraphPad Software, Inc., San Diego, CA, USA). One-way analysis of variance (ANOVA) was employed to compare differences among multiple groups, and two-way ANOVA was utilized for pairwise comparisons. A P-value of less than 0.05 was considered statistically significant. The following significance levels are indicated in the figures: *P < 0.05, **P < 0.01, and **P  < 0.001.

Results

VK4 inhibits leukemia cells proliferation

After a broad screening for anti-leukemia compounds (Fig. S1), VK4 (Fig. 1A) was found to be a potential candidate. As data shown in Fig. 1B ~ D, the treatment of VK4 for 24 h significantly inhibited the proliferation of HL60, Jurkat, and K562 cells, with IC₅₀ values of VK4 against these three leukemia cells at 5.35 µM, 8.44 µM, and 11.59 µM, respectively (Fig. 1F). However, VK4 did not affect the proliferation of healthy PBMC even under the concentration of 40 µM (Fig. 1E). Moreover, the anti-leukemia effects were also evaluated in the primary leukemia cells isolated from the bone marrow blood in clinical leukemia patients (Table 1). The CCK-8 results indicated that VK4 could inhibit cell proliferation in primary leukemia cells, with the IC₅₀ values from 3.69 µM to 9.39 µM (Fig. S2). Altogether, these data indicated that VK4 exhibited an excellent anti-leukemia activity both in leukemia cell lines and primary leukemia cells.

Fig. 1.

Effect of VK4 on the proliferative activity in leukemia cells and healthy PBMC. (A) The chemical structural formula of VK4; (B) HL60 cells were treated with 0, 2, 3, 4, 5, 6, 7, and 8 µM VK4, and Jurkat (C), K562 (D) and human healthy PBMCs (E) cells were treated with VK4 at the concentration of 0, 0.625, 1.25, 2.5, 5, 10, and 20 µM for 24 h. Afterwards, the cell viability was detected by CCK-8 kits; (F) The IC₅₀ values of VK4 on cell proliferation were presented as mean ± SD (n = 3)

Table 1.

The IC₅₀ value of VK4 against primary leukemia cells

Clinical sample	FAB Classification	Source	IC50 (µM)
1	B-ALL	BM	5.99
2	AML	BM	3.69
3	B-ALL	BM	7.18
4	CML	BM	6.36
5	B-ALL	BM	8.95
6	T-ALL	BM	9.39

VK4 induces cell cycle arrest in leukemia cells

Cell proliferation is controlled by the progression of cell cycle. To explore whether VK4 inhibited the leukemia proliferation through inducing cell cycle arrest, we employed flow cytometry with PI staining to examine the cell cycle distribution. As data shown in Fig. 2A ~ D, VK4 could induce HL60 and Jurkat cells arresting in the S-phase in a dose-dependent manner; while K562 cells were significantly arrested in the G2/M phase by VK4 (Fig. 2E and F). Intriguingly, VK4 had no effect on the cell cycle arrest in primary B-ALL cells from patients 1 (Fig. 2G and H). The progression of mammalian cell cycle is driven by the regulatory network consisting of cyclins and cyclin-dependent kinases (CDKs), next, we examined the expression levels of these proteins. Results from western blotting found that VK4 could downregulate the expression of CDK1, CDK2, Cyclin A2, and Cyclin B1 in HL60 cells (Fig. 2I and L), as well as Cyclin E2 and CDK4 in Jurkat cells (Fig. 2J and M). Consistent with the flow cytometry results with the arresting of G2/M phase in K562 cells, VK4 significantly reduced the expression levels of Cyclin A2, Cyclin B1, CDK4 and CDK1 (Fig. 2K and N). Collectively, these data suggested that VK4 triggered cell cycle arrest in leukemia cells and downregulated the expression levels of corresponding cyclin proteins in a dose-dependent manner.

Fig. 2.

VK4 induces cell cycle arrest in leukemia cells. (A) HL60 cells, Jurkat cells (B), K562 cells (C) and primary B-ALL cells (D) were treated with the indicated concentration of VK4 for 12 h, then cells were stained with PI and subjected to flow cytometry analysis. (E−H) Quantification of all phases of the cell cycle from (A−D). (I) Western blotting was used to detect the expression level of intracellular cyclins in HL60 cells, Jurkat cells (J), and K562 cells (K) after the treatment of VK4 at the indicated concentrations for 12 h. (L−N) Quantitative analysis of protein expression from (I−K) by Image J. P value < 0.05 indicates a statistically significant difference. “ns” denotes no statistical significance, * P < 0.05, ** P < 0.005, *** P < 0.001 (Vehicle group (0 µM) vs. VK4 treatment group, n = 3)

VK4 induces apoptosis in leukemia cells

To further identify whether the anti-proliferative effects of VK4 on leukemia cells were associated with the induction of apoptosis, the flow cytometry by Annexin V-Alexa Fluor 488/PI double-staining was used to analyze the apoptotic cells. As shown in Fig. 3A, the treatment of VK4 at the indicated concentrations for 24 h mainly induced the late apoptosis (in the upper right quadrants of the dot plot) in HL60, Jurkat, and K562 cells, especially in the highest concentration, the apoptotic cells accounted for 86.4%, 47.0%, and 50.33% in HL60, Jurkat, and K562 cells, respectively. Meanwhile, we also evaluated the antit-leukemia activity of VK4 against another AML cell lines, including Kasumi-1, KG-1α and THP-1. It was found that VK4 also exhibited potent cytotoxic activity in these AML cells (Fig. S4A, S4B), and induced cell apoptosis (Fig. S4C–G).

Fig. 3.

VK4 induces apoptosis in leukemia cells. (A) HL60, Jurkat and K562 cells were treated with the indicated concentration of VK4 for 24 h, and then cells were analyzed by Annexin V-Alexa Fluor 488/PI staining and detected by flow cytometry, with data analyzed and plotted using Flowjo 10.8.1 and quantified using GraphPad Prism 10.0. * P < 0.05, ** P < 0.005, *** P < 0.001 (Vehicle group (0 µM) vs. VK4 treatment group, n = 3). (B) HL60, Jurkat (C) and K562 (D) cells were treated with VK4 at the indicated concentration for 24 h, and the expression level of Caspase 3 and Cleaved-Caspase 3 were measured by western blotting, GAPDH was employed as an internal loading control. (E) Quantitative analysis of protein expression from B ~ D by Image J. P value < 0.05 indicates a statistically significant difference. “ns” denotes no statistical significance, * P < 0.05, ** P < 0.005, *** P < 0.001 (Vehicle group (0 µM) vs. VK4 treatment group, n = 3). (F) Primary B-ALL cells were treated with the indicated concentration of VK4 for 24 h, and the expression level of pro-Caspase 3, Cleaved-Caspase 3, PARP and Cleaved-PARP were detected by western blotting. β-Actin was employed as an internal loading control. (G) Quantitative analysis of protein expression from (F) by Image J. P value < 0.05 indicates a statistically significant difference. “ns” denotes no statistical significance, * P < 0.05, ** P < 0.005, *** P < 0.001 (Vehicle group (0 µM) vs. VK4 treatment group, n = 3)

Cysteinyl aspartate specific proteinase 3 (Caspase 3) is the central executer of apoptosis with poly ADP-ribosepolymerase (PARP) as its first substrate [36]. Results from western blotting demonstrated that VK4 could significantly increase the activation of caspase 3 in HL60, Jurkat, and K562 cells (Fig. 3B–E). Moreover, in primary B-ALL cells, VK4 was found to promote the activation of Caspase 3 and the cleavage level of PARP in a dose-dependent manner (Fig. 3F and G). Taken together, these data indicated that VK4 was capable to induce leukemia cell apoptosis.

VK4 induces apoptosis in HL60 and K562 Cells through the mitochondrial pathway

Apoptosis is one type of program cell death and plays an important role in killing damaged cells and preventing transformed cells growth through various caspases. According to the implicated death signals, apoptosis can be divided into two main types: Caspase 9-mediated intrinsic pathway and Caspase 8-mediated extrinsic pathway. To determine which pathway was involved in induction of apoptosis caused by VK4, western blotting was applied to detected the activation of signals associated with the apoptotic pathway. As shown in Fig. 4A ~ C, upon the treatment of VK4 for 24 h, Caspase 9 was notably activated in HL60, Jurkat and K562 cells, while Caspase 8 was only activated in Jurkat cells, indicating that the intrinsic apoptosis was induced by VK4 in all these three leukemia cell lines. And extrinsic pathway might also be involved in the process of apoptosis in Jurkat cells caused by VK4. The intrinsic pathway is also known as mitochondrial pathway which is charactered by the increased permeability of the outer mitochondrial membrane and release of cytochrome c (Cyt C) resulting from activation of BCL-2 family members. Therefore, some key proteins associated with the intrinsic pathway were also detected by western blotting. It was shown that VK4 could notably increase the protein level of Cyt C and reduce the expression level of pro-apoptotic proteins including XIAP, Survivin, Bcl-xL, and Bcl-2, while BAD and BAX were not affected by VK4 (Fig. 4A−C, Fig. S3).

Fig. 4.

VK4 induces apoptosis in leukemia cells through the mitochondrial pathway. (A) HL60 cells were treated with VK4 at the concentration of 4.0, 6.0, and 8.0 µM for 24 h, K562 cells (B) with 10.0, 12.5, and 15.0 µM for 24 h, and Jurkat cells (C) with 5.0, 7.5, and 10.0 µM for 24 h. The expression of pro-apoptotic proteins, anti-apoptotic proteins, and caspase family proteins were measured by western blotting, (D) Changes of cellular MMP was probed by JC-1 staining and flow cytometry. Data were analyzed and plotted using Flowjo 10.8.1. The column chart shows the ratio of JC-1 monomers to JC-1 aggregates (n = 3). “ns” denotes no statistical significance, * P < 0.05, ** P < 0.005, *** P < 0.001 (Vehicle group (0 µM) vs. VK4 treatment group, n = 3)

During the mitochondrial apoptosis, the loss of mitochondrial membrane potential (MMP, Δψ_m) is demonstrated to be an early event [37]. To determine whether the MMP was reduced under the treatment of VK4, the cyanine dye JC-1 was used to discriminate the energized and deenergized mitochondria [38]. When JC-1 aggregates in the mitochondrial matrix, it will emit red fluorescence in healthy cells; while the JC-1 monomer will emit green fluorescence when MMP declines in damaged cells [39]. Results from flow cytometry demonstrated that the treatment of VK4 for 24 h could significantly increase the percentage of JC-1 monomers in a dose-dependent manner in HL60, Jurkat and K562 cells, indicating a reduction of intracellular MMP (Fig. 4D). Taken together, these data suggested that VK4 could induce the mitochondrial apoptosis to exert its anti-leukemia effects.

VK4 exerts anti-leukemia effects by regulating the JAK/STAT signaling pathway

The JAK/STAT signaling pathway plays crucial roles in maintaining body healthy, while dysregulation of this pathway is associated with the development of various cancers [40]. STAT3 and STAT5 are vital members of the STAT family that are widely reported to be implicated in the regulation of mitochondrial functions and cellular energy metabolism, and the hyperactivation of them usually stimulates the proliferation of malignant cells [7]. It was reported that the JAK2-STAT3/5 signaling pathway was excessively activated in leukemia [41]. We next checked whether the treatment of VK4 reduce the activation of JAK/STAT signaling pathway. It was found that upon the treatment of VK4, the phosphorylated forms of JAK2, STAT3, and STAT5 were obviously down-regulated in HL60, K562 (Fig. 5A, S7) and Jurkat cells (Fig. S5A, S5B), suggesting that VK4 could inhibit the JAK/STAT signaling pathway in leukemia.

Fig. 5.

VK4 exerts anti-leukemia effects through the JAK/STAT signaling pathway. (A) HL60 cells were treated with 4.0, 6.0, and 8.0 µM VK4 for 24 h, and K562 cells were treated with 10.0, 12.5, and 15.0 µM for 24 h. Protein expression level of JAK2, p-JAK2, STAT3, p-STAT3, STAT5, and p-STAT5 were measured by western blotting. (B) Real-time kinetic binding parameter of VK4 interacting with JAK2 based on BLI. (C) HL60 cells were pretreated with 15 µM AG490 or not for 12 h, and then treated with VK4 at the indicated concentration for another 24 h. Cell viability was measured by CCK-8 assay kit. (D) K562 cells were pretreated with 10 µM AG490 or not for 12 h, and then treated with VK4 at the indicated concentration for another 24 h. Cell viability was measured by CCK-8 assay kit. * P < 0.05, ** P < 0.005, *** P < 0.001 (VK treatment group vs. VK4 + AG490 treatment group, n = 3). (E) HL60 cells were pretreated with 15 µM AG490 or not for 12 h, and then treated with 8 µM VK4 for another 24 h; K562 cells were pretreated with 10 µM AG490 or not for 12 h, and then treated with 15 µM VK4 for another 24 h, and then cells were analyzed by Annexin V-Alexa Fluor 488/PI staining and detected by flow cytometry, with data analyzed and plotted using Flowjo 10.8.1 and quantified using GraphPad Prism 10.0. “ns” denotes no statistical significance, * P < 0.05, ** P < 0.005, *** P < 0.001. (F) HL60 and K562 cells were treated as (E), and then apoptosis-associated proteins were measured by western blotting

To further identify whether JAK2 and STAT3/5 were the targets for VK4 that contributed to the inhibition of leukemia cells proliferation, we performed the Bio-Layer Interferometry (BLI) assay (Octet R8, Sartorius). As shown in Fig. 5B, VK4 had a direct interaction with JAK2 (KD = 1.018 × 10^− 6), but no interaction signals were observed between VK4 and STAT3 or STAT5 (data not shown). Besides, a special inhibitor for JAK2/3 and STAT3, AG490 was used to determine whether the anti-leukemia effects of VK4 were depended on the JAK/STAT signaling pathway. It was found that, a treatment of AG490 alone under the concentration of 40 µM had no effects on the proliferation of HL60, Jurkat and K562 cells (Fig. S6); however, a pretreatment of 15 µM or 10 µM AG490 for 12 h could partially recuse the cytotoxic activity of VK4 in HL60 and K562 cells, respectively (Fig. 5C and D), but the restoration effect was not observed in Jurkat cells (Fig. S5C); besides, data from the flow cytometry assay also found AG490 could antagonize VK4-mediated apoptosis in HL60 and K562 cells (Fig. 5E), which was not observed in Jurkat cells (Fig. S5D, S5E). Correspondingly, results from western blotting also demonstrated that, compared with the vehicle group, the alone treatment of AG490 had no significant effects on the manifestation of proteolytically-cleaved Caspase 3, Caspase 9 and PARP; while the pretreatment of AG490 could noticeably decrease the expression levels of cleaved-Caspase 3, -Caspase 9 and -PARP in HL60 and K562 cells (Fig. 5F, S7), which were not observed in Jurkat cells (Fig. S5F, S5G). All together, these data suggested the inhibition of JAK/STAT signaling pathway by VK4 might contribute to its anti-leukemia effects in HL60 and K562 cells.

VK4 inhibits the leukemia cells proliferation in human acute myeloid leukemia xenografts mice model

The HL60 xenografts model was established in BALB/c nude mice by tail vein injection to assess the anti-leukemia effects of VK4 in vivo (Fig. 6A). Blood smear test by Wright-Giemsa Staining was used to examine the numbers of human leukemias in the peripheral from mice tail vein every 7 days. Approximately 14 days post HL60 engraftment, HL60 cells were found to be successfully colonized in vivo, mice were randomized into four groups and treated daily by tail vein injection with vehicle control or VK4 at three dosages as following: 2.5 mg/kg, 5.0 mg/kg, and 10.0 mg/kg for 28 days. Compared with the vehicle control group, the treatment of VK4 displayed no significant adverse effect on mice body weight (Fig. 6B), on the contrary, VK4 treatment could significantly accelerate the weight gain in the middle and high-dose groups (Fig. 6B−C), indicating the safety of VK4 in vivo. Moreover, compared with the vehicle control group, treatment with VK4 at doses of 5 mg/kg and 10 mg/kg significantly prolonged the survival of mice (Fig. 6D).

Fig. 6.

VK4 inhibits leukemia cells proliferation in vivo. (A) Flowchart for establishment of HL60 xenografts model in BALB/c nude mice. (B) The individual body weight of mice was recorded at the indicated days and presented as mean ± SD (Vehicle control group and 2.5 mg/kg VK4 treatment group: n = 8; 5 mg/kg and 10 mg/kg VK4 treatment group: n = 10). “ns” denotes no statistical significance, *** P < 0.001 (Vehicle group (0 µM, n = 8) vs. VK4 treatment group (2.5 mg/kg n = 8; 5, 10 mg/kg, n = 10). (C) Mice were sacrificed 28 days post VK4 treatment. (D) Survival of mice administered different doses of VK4 or vehicle control (n = 10 per group) was analyzed using the Kaplan–Meier method. The survival curves were compared using a Log-rank (Mantal-Cox) test (2.5 mg/kg vs. control, ns (no significance); 5 mg/kg vs. control, P = 0.031, Hazard Ratio (log rank = 0.222); 10 mg/kg vs. control, P = 0.0098, Hazard Ratio (log rank = 0.111)). (E) At the end of treatment, peripheral venous blood from the mice tail vein was collected and applied to blood smear test and Wright-Giemsa staining. Leukemia cells were observed by Zeiss microscope. (F) Quantification of leukemia cells were from (E). ** P < 0.005, *** P < 0.001 (control group vs. VK4 treatment group, n = 3). (G) HE staining of the mice liver and kidney tissues at the end of treatment. (H) Immunohistochemical results from CD33 staining in the mice spleen at the end of treatment

Data from the peripheral blood smears assay found that, compared with the vehicle control group, VK4 could inhibit the proliferation of HL60 cells in a dose-dependent manner in vivo (Fig. 6E and F). Moreover, the side effects on liver and kidney were also evaluated, and no obvious organ damages were observed from the data of HE staining in VK4 treated groups (Fig. 6G). In addition, the human leukemia cells infiltration in the mice spleen was detected by immunohistochemistry through staining human CD33, which was reported to be highly and specifically expressed in HL60 cells [42]. As shown in Fig. 6H, compared with the vehicle control group, the CD33 positive cells were reduced in VK4 treated mice in a dose-dependent manner, suggesting that VK4 could inhibit the infiltration of leukemia cells in the spleen in vivo. Collectively, these data demonstrated that VK4 displayed an anti-leukemia effect in vivo without significant toxic effects, which was in accordance with the findings in vitro.

Discussion

Leukemia is a progressive disease of the blood-forming organs and ranks in the top 10 of cancers in mortality globally [3]. According to the leukocyte differentiation and the cellular origins of leukemia, it can be mainly categorized into lymphoid and myeloid leukemia, with myeloid leukemias being more aggressive and poor responses to intensive chemotherapy [43]. Natural products exhibit good activities for the development of anti-cancers drugs. In previous studies, VK4 represented potential anti-tumor activities in solid tumors [25, 26], but its effects in anti-leukemia was not explored. In this study, we found that VK4 could inhibit the leukemia viability both in vitro and in vivo. Flow cytometry assay revealed that VK4 induced the cell cycle arrest at S phase in HL60 and Jurkat cells, and G2/M phase in K562 cells; meanwhile, apoptosis was also induced in HL60, Jurkat and K562 cells, as well as in primary B-ALL cells. As known, apoptosis is one type of programmed cell death and is initiated by the activation of Caspase 3 [36]. The apoptotic pathway is typically categorized into exogenous and endogenous mitochondrial apoptosis [44]. The mitochondrial apoptosis is driven by numerous stimuli, like DNA damage, hypoxia, and metabolic stress etc. to stimulate the release of caspases activators from mitochondria. A reduction of MMP is widely considered as an indicator of the initiation of mitochondrial apoptosis [45], with Caspase 9 being an essential initiator in the apoptotic pathway [46]. The Bcl-2 family proteins are involved in the mitochondrial apoptotic pathway. Apoptosis-promoting proteins like Bcl-xL and Bcl-2 predominantly reside on the mitochondrial membrane, while pro-apoptotic proteins such as Bad and Bax mainly found in the cytoplasm. Onset of apoptosis, Bax oligomerization forms pores and permeabilizes the mitochondrial outer membrane, leading to a decrease in MMP. Subsequently, the permeability of the mitochondrial membrane rises, and apoptogenic factors within the mitochondria (for instance Cytochrome C (Cyt C)) are discharged into the cytoplasm. Once Cyt C enters the intracellular space, it enters an interaction with Apaf-1 to form an apoptotic complex. The apoptosome then recruits and activates Pro-Caspase 9, leading to the formation of the Caspase 9 holoenzyme. The Caspase 9 takes on the crucial task of activating effector Caspase, initiating the Caspase cascade reaction, which cleaves more than 100 substrates in the cell (such as PARP), ultimately leading to cell apoptosis [47]. In our study, we discovered that the VK4 treatment for 24 h significantly reduced MMP in the three leukemia cell lines (Fig. 4D). Western blotting results also showed that in HL60, Jurkat, and K562 cells, the treatment of VK4 could remarkably elevate the levels of activated Caspase 9, Caspase 3, and PARP, while downregulating the expression level of Bcl-xL and Bcl-2 (Fig. 4A−C). In addition, we determined the protein expression level of Caspase 8, a key protein molecule in the extrinsic apoptotic pathway. The results indicated that a significant increase of Cleaved-Caspase 8 was only observed in Jurkat cells, while no significant changes in HL60 and K562 cells were observed. Survivin is a critical apoptosis-inhibitory protein that is benefited for cell survival. Survivin is usually highly expressed in cancers, and exerts its anti-apoptosis effects by downregulating Caspases or interacting with other apoptosis regulators like XIAP [48, 49]. The results from western blotting in our studies demonstrated that VK4 could remarkably reduce the expressions of Survivin and XIAP (Fig. 4A−C). These findings collectively implied that the endogenous apoptotic pathway triggered by VK4 played a vital role in its anti-leukemia effects.

The JAK/STAT signal transduction pathway has been demonstrated to exert a vital part in the biological functions of cells, encompassing cell proliferation, apoptosis and differentiation etc. The abnormal activation and mutations of JAK/STAT signals are usually related to the genesis and progression of cancer [50]. It was reported that in leukemia and other hematological tumors, the JAK/STAT signaling is usually excessively activated, especially with hyperphosphorylated JAK2, STAT3, and STAT5 [51]. In our study, high expression of phosphorylated JAK2, STAT3, and STAT5 were found in untreated leukemia cells (Fig. 5A), which were consistent with the previous reports. The treatment of VK4 could downregulate the phosphorylation level of JAK2, STAT3, and STAT5 in the JAK/STAT signaling pathway; however, the pretreatment of JAK2/3 and STAT3 inhibitor AG490 could partially counteract the effects of VK4 in HL60 and K562 cells in cell proliferation and cell apoptosis (Fig. 5C−D). Data from BLI also confirmed a direct interaction between VK4 and JAK2 (Fig. 5B), but no interaction between VK4 and STAT3/5 was observed. We speculated that JAK2 was a potential target for VK4, and the inhibition of JAK2 by VK4 facilitated its anti-leukemia activity in HL60 and K562 cells. When JAK2 was inhibited by AG490, VK4 would lose its target in HL60 and K562 cells, hence, the anti-leukemia activity triggered by VK4 could be rescued. Although the application of AG490 alone had no inhibitory effect on HL60, K562, and Jurkat cells (Fig. S6), interestingly, it could promote the process of apoptosis in Jurkat cells induced by VK4 (Fig. S5D), indicating that the JAK/STAT pathway might not be involved in the anti-tumor effects generated by VK4 in Jurkat cells. We hypothesized that VK4 might apply different mechanisms to inhibit lymphoid and myeloid leukemia. A further investigation is required to uncover the underlying mechanisms involved in the anti-tumor effects generated by VK4 in Jurkat cells.

Furthermore, we also established a HL60 xenograft nude mice model to explore the anti-leukemia effect of VK4 in vivo. It was found that VK4 could inhibit leukemia cells proliferation in vivo at the concentration rang of 2.5−10 mg/kg. And no obvious adverse effects in body weight, as well as in liver and kidney tissues were observed. Comprehensively, VK4 exhibited excellent cytotoxic activities against leukemia cells through inhibiting the JAK/STAT signaling pathway. The in vitro and in vivo anti-leukemia effects suggested VK4 as a promising candidate for cancer therapeutics in leukemia by target JAK2-STAT3/5 signaling pathway.

Author contributions

Xiuyu Li and Pengcheng Zhu, Investigation and Writing-Original draft preparation. Shaowen Hu, Investigation. Qing Huang, Data Curation. Yilin Qin, Investigation. Huifang Zhu, Funding acquisition, Writing-Reviewing and Editing.

Funding

This study was supported by grants from the Natural Science Foundation of Jiangxi Province (20232ACB206017, 20212BCJL23049) and the Program of Science and Technology Guided plan of Ganzhou (2025YLCE0096).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

This study complied with the Declaration of Helsinki and adhered to animal experimental ethics guidelines, and the collection of related blood samples had been approved by the Research Ethics Committee of the First Affiliated Hospital of Gannan Medical University (Ethics No. LLSC-2024 No. 283). Animal experimental procedures were carried out in strict compliance with the ethical standards established by the Animal Ethics Committee of the First Affiliated Hospital of Gannan Medical University (Ethics No. LLSC-2024 No. 078).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical trial number

Not applicable.