Casticin inhibits the hedgehog signaling and leads to apoptosis in AML stem-like KG1a and mature KG1 cells

Tuğba Erkmen Doğru; Gizem Gülfidan; Halil Ateş; Kazım Yalçın Arga; Semra Koçtürk

doi:10.1515/tjb-2024-0149

Article Open Access

Casticin inhibits the hedgehog signaling and leads to apoptosis in AML stem-like KG1a and mature KG1 cells

Tuğba Erkmen Doğru , Gizem Gülfidan , Halil Ateş , Kazım Yalçın Arga and Semra Koçtürk

Published/Copyright: December 3, 2024

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From the journal Turkish Journal of Biochemistry Volume 50 Issue 1

Abstract

Objectives

Acute myeloid leukemia (AML) is a severe blood cancer with less than 50 % long-term survival. Despite advancements in treatment options, relapse is still the major obstacle. The main reason of this problem is ineffective targeting of leukemic stem cells (LSCs), which play an important role in tumor development and relapse. In our previous studies, we found that casticin, the major polyphenolic component of Vitex trifolia’s fruit, targets both leukemic cells and LSCs without affecting healthy tissues. Therefore, in this study, we aimed to investigate the effect of casticin-mediated cell death in relation to the LSCs-favored survival pathways at gene and protein expression levels using in vitro LSC-like and parental leukemic cell models.

Methods

We validated the LSC character of KG1a and KG1 cells (84.55 % CD34+, CD38- and 93.55 % CD34+, CD38+, respectively) by flow cytometry. For the investigation of casticin’s mechanism of action, we employed real time-PCR, western blotting and bioinformatics analyses.

Results

Our results showed an increase in cleaved PARP/β-actin ratio but no change in LC3BI/II and SQSTM/β-actin ratios. Our gene expression, bioinformatics and immunoblotting analyses represented significant decrease in Shh, Gli and Wnt levels. We also elucidated a possible crosstalk between Hedgehog and other oncogenic cascades via the Gli, Notch, YAP, p38, Mcl-1, and Myc proteins in casticin mediated anti-leukemic effect.

Conclusions

In conclusion, we found that casticin induces apoptosis in both LSC-like and parental leukemia cells mainly by suppressing Shh signaling, which is crucial for LSC survival and AML relapse.

Keywords: acute myeloid leukemia; apoptosis; cancer stem cells; casticin; cell signaling

Introduction

Acute myeloid leukemia (AML) is the most common form of acute leukemia in children. Standard treatment for AML dates back to the 1970s, a regimen that disrupts DNA and RNA synthesis in all rapidly dividing cells of the body [1]. Conventional chemotherapies have some limitations in AML treatment due to their cytotoxic effects on healthy tissues and inability to target leukemia stem cells [2], [3], [4]. Although the U.S. Food and Drug Administration (FDA) has approved new therapeutic agents for AML treatment, their effectiveness is still limited with less than 50 % 5-year survival rates [5], [6].

Leukemic stem cells (LSCs) represent a small fraction (only 1–4 %) of the total AML cells, which can self-regenerate and have unlimited re-population capacity. Therefore, LSCs have the ability to initiate leukemia, and insufficient eradication of these cells leads to drug resistance and relapse [7], [8]. Hence, it is important to incorporate LSC-targeted agents into therapeutic strategies.

Natural products have been a promising source for anti-cancer drug discovery for decades [9]. Phytochemicals have the ability to induce specifically cancer cell death by affecting multiple oncogenic signaling pathways [10]. Since survival signaling pathways like Notch, Wnt, Hedgehog, and Hippo cascades are critical for targeting LSCs [11], [12], [13], [14], [15], phytochemicals may be a promising source to disrupt these signals, leading to programmed cell death in both LSCs and leukemic blasts for ultimate AML treatment [16], 17].

Casticin (3′, 5-dihydroxy-3, 4′, 6, 7-tetramethoxyflavone) is a predominant polyphenolic constituent of the fruit of Vitex trifola L [18]. and studies reported that casticin induces apoptosis in AML cells [19], [20], [21]. In particular, Righeschi et al. reported that casticin has the most potent anti-proliferative effect on lymphoblastic leukemia cells among the well-known polyphenols [22]. In our previous study, we showed that 2 μM casticin induces apoptosis in both leukemic cells and LSCs without affecting healthy cells [23]. Although the anti-proliferative effect of casticin on leukemia cell lines is known, the mechanism of action of casticin is still unelucidated. Hence, this study aimed to uncover the underlying molecular mechanism focusing on signaling pathways associated with casticin-induced cell death in LSC-like KG1a and AML blast model KG1 cell lines at gene and protein expression levels.

Materials and methods

Cell culture

KG1 and KG1a cells were obtained from ATCC (ATCC, Rockville, MD, USA). KG1a and KG1 cells cultured in 20 % fetal bovine serum (Biochrom, Germany) involved Iscove’s Modified Dulbecco’s Medium (IMDM) (ATCC, 30–2005) at 37 °C in 5 % CO₂ incubator. KG1a cell line consists of stem-like AML cells that lack of differentiation capacity. However, KG1 cell line, which involves a more mature AML cells, exhibits differentiation capability into macrophages [24].

Cell viability assays

Casticin (≥98 % purity, Sigma, MO, USA) was dissolved in dimethyl sulfoxide (DMSO; AppliChem, Germany) with a stock concentration of 50 mM. Further dilutions were made with fresh culture medium, maintaining DMSO concentration<0.1 %. Cell viability and IC50 values of casticin were assessed using MTT colorimetric assay. In brief, 50 µL of cell suspension was seeded into a 96-well plate, after overnight starvation, cells were treated with casticin for 24 or 48 h, then incubated with 10 µL/well MTT solution (5 mg/mL). After solubilization, absorbance at 562 nm was measured by using microplate reader Biotek (ELX800, USA).

Phenotypes of the cell lines

To validate our in vitro stem cell model, flow cytometry was used to assess CD34+ and CD38-expression in KG1a and KG1cell lines. Cells (5 × 10⁵–1 × 10⁶) were washed and treated with CD34- FTC (Invitrogen) and CD38-APC (Invitrogen) antibodies, then analyzed using Navios Flow Cytometer (Beckman Coulter, USA).

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis

To explore the mechanism of action of casticin treatment, RT-qPCR analyses were performed using gene panel (IDT, 14246249) comprising 47 genes, including 42 genes involved in cell survival and apoptotic signaling along with 5 housekeeping (Table 1). Briefly, after the treatment the total RNA was extracted using High Pure RNA Isolation Kit (Roche) followed by cDNA synthesis using EvoScript Universal cDNA Master (Roche). PCR amplification conditions included pre-denaturation (95 °C) for 10 s, denaturation (95 °C) for 10 min, annealing and extension for 30 s at 60 °C, over 45 cycles. The RT-qPCR was performed with Roche Light Cycler 480 Real-time PCR system by using FastStart Essential DNA Green Master Mix (Roche) in 96-well plates. For data normalization, the mean ∆CT value of five housekeeping genes was used and the data were analyzed with the 2^−∆∆Ct method [25]. Fold change cut off was set at -fold and higher to identify upregulated and downregulated genes. For the visualization of all gene expression changes, heat maps were created by using package ‘heatmap’ version 1.0.12 in R (v.4.2.2).

Table 1:

Genes employed in the panel.

Pathway/phenotype	Genes
Drug resistance	ABCB1, ABCC1, ABCG2
Seramid formation pathway	CERS, NSMAF, UGCG
PI3K/Akt/mTOR pathway	AKT1, mTORC1, PIK3CB, PIK3CG, PTEN, PTPN11
Ras/MEK/ERK	ERK1, JNK1, KRAS, NRAS, MEKK1, P38 (MAPK14)
NFKB	P65, HSP90
Notch	NOTCH1
Hedgehog	GLI1, SHH
Hippo	ALDH1A1, ALDH3A1, YAP/TAZ
Apoptosis	BAD, BAX, BCL-2, BCL-XL, MCL-1, MYC, PARP1, P53
Autophagy	BECN1 (Beclin), LC3B, SQSTM1
Wnt/Beta-catenin	CTNNB1 (Beta-catenin), LEF, WNT1
Jak/Stat	STAT3, STAT5
Housekeeping	ACTB (Beta-actin), B2M, RPL37A, SRP14, UBE2D2

Gene overrepresentation analysis

In order to determine the biological processes and signaling pathways related to cell death, the Consensuspathdb tool were used in gene set overrepresentation analyses [26]. Gene Ontology (GO) annotations [27] were utilized to provide information on molecular functions and biological processes, while the Kyoto Encyclopedia of Genes and Genomes (KEGG) [28] and Reactome [29] databases served as valuable resources for signaling and metabolic pathway information. Statistical significance was determined using Fisher’s exact test, and the Benjamini-Hochberg correction was applied to adjust the p-values. Results with adjusted p-value ≤ 0.05 were considered statistically significant.

Protein-protein interaction analysis

A protein-protein interaction network was constructed to depict the connections among the proteins encoded by the significantly altered genes, as well as other proteins involved in relevant biological processes. Establishment of this network was achieved by using human physical protein-protein interaction data (version 4.4.206) from the Biological General Repository for Interaction Datasets (BioGRID) and Cytoscape software facilitated network visualization [29], 30].

Assessment of protein expressions

Significantly altered gene expressions, as identified by RT-qPCR and bioinformatics, were also assessed at protein expression level by using western blotting. Briefly, whole-cell lysates were prepared using RIPA buffer (Thermo, MA, USA). Subcellular fractionation was performed using the NEPER kit (Thermo, MA, USA). Protein concentrations were detected by BCA assay (Pierce Chemical, USA) with approximately 40 ug of total protein was loaded per well. Following SDS-PAGE, proteins were transferred to PVDF membranes by semi-dry or wet-transfer methods. Membranes were blocked with 5 % BSA (Biochrom, Germany) in Tris-buffered saline containing 0.1 % Tween 20 (Sigma, St. Louis, MO, USA) and immunoblotted overnight at 4 °C with primary antibody (PARP, LC3BI/II, SQSTM1/p62, Sonic hedgehog (Shh), Gli, Wnt) followed by 1 h incubation with the horseradish peroxidase-linked secondary antibody (Goat anti-rabbit IgG-HRP; Goat anti-mouse IgG-HRP, Cell signaling). Protein detection was carried out using the West Pico chemiluminescent substrate kit (Thermo, MA, USA).

Statistical analysis

All experiments were conducted three times and the results are expressed as mean±SEM. Statistical significance was determined using one-way-ANOVA in SPSS version 22.

Results

Phenotypes of the cell lines

According to our results, approximately 84.55 % of KG1a cells exhibited the CD34+, CD38-phenotype, while 93.55 % of KG1 cells demonstrated the CD34+, CD38+ phenotype (Figure 1). Since CD34+ and CD38-phenotype is accepted as the characteristic feature of AML LSCs [7], we used the KG1a cell line as an in vitro model for LSCs and the KG1 cell line as their parental cell line to represent mature leukemic cells.

Figure 1:

Flow cytometry diagrams and graph of CD34 and CD38 expression in KG1a and KG1cell lines.

mRNA expressions of signaling pathways

All genes in the designed panel were categorized according to relevant biological processes, molecular pathways, and significant expression changes for KG1a, KG1 cells (Supplementary Figures S1 and S2).

The changes in gene expression were visualized by a heat map, which allowed us to comparatively evaluate the effects of casticin treatment on target genes in the KG1a and KG1 cell lines (Figure 2). RT-qPCR results showed that treatment of KG1a cells with casticin resulted in a significant decrease in the expression of SHH, GLI, and WNT mRNA levels, genes known to play an important role in LSC survival [4]. An increase in the expression of P53 and P38 genes, which are closely associated with apoptosis, was detected. In addition, a significant increase was found in the expression of genes that support the survival of leukemic cells and LSCs, such as ABCC1, ABCG2, MCL-1, NOTCH1, NRAS, and ERK. In addition, treatment with casticin resulted in a significant increase in the expression of ALDH1A1, which has high expression and enzymatic activity in LSCs (Figure S1 and Figure 2A). In KG1 cells, casticin treatment significantly decreased the expressions of SHH, GLI, and WNT genes. In addition, there was also a significant decrease in the gene expression of ABCB1, which encodes p-glycoprotein that plays a role in drug resistance (Figure S2 and Figure 2A). After casticin treatment, a significant decrease in the mRNA levels of SHH, GLI, and WNT was detected in both cell lines. While there was a significant decrease in ABCB1 mRNA expression in KG1 cells following casticin treatment, there was no significant alteration in KG1a cells. In addition, although casticin caused very significant increase in ALDH1A1 mRNA levels in KG1a cells, the treatment did not affect ALDH1A1 mRNA levels in KG1cells (Figure 2B).

Figure 2:

Heat map representing the mRNA expression changes in casticin-treated KG1a and KG1 cell lines. (A) Fold changes in 42 genes in response to casticin treatment (compared to their respective non-treated control groups). (B) Differentially expressed genes with at least 2-fold changes in response to casticin treatment (compared to their respective non-treated control groups). The blue color indicates a decrease in expression, while the red color indicates an increase in expression.

We also analyzed cell signaling pathways-associated gene alterations and pathway-pathway interactions considering differentially expressed genes (by p-value as ≤0.05 and fold change as ≥2) in response to the casticin treatment (Figure 3).

Figure 3:

Cell signaling pathways-associated gene alterations and pathway-pathway interactions.

In evaluating the effects of casticin on cell survival signaling pathways, it was appropriate to focus on Shh and Wnt/β-catenin signaling pathways in both cell lines. To reveal the functional relationships between the proteins and their potential roles in casticin-mediated cell death, a protein-protein interaction network (PPI) was constructed (Figure 4). According to the PPI network and RT-qPCR results, the most interactive proteins were found to be Gli, Shh, and Wnt.

Figure 4:

Protein-protein interaction (PPI) network depicting the interactions between proteins encoded by genes that show significant changes (fold change of 2 or higher) in RT-qPCR analysis. The blue nodes represent the proteins included in our RT-qPCR gene panel, while the others represent other proteins that could potentially interact with them.

Protein expression levels

Confirmation of apoptotic cell death was achieved through the analysis of PARP protein levels. The ratio of cleaved PARP/β-actin was significantly increased in casticin-treated KG1a and KG1 cells compared with the corresponding control groups (p<0.01 for both KG1a and KG1) (Figure 5A and B). The findings were consistent with the flow cytometry results [23] and it was confirmed that 2 μM casticin treatment induced apoptotic cell death in both cell lines.

Figure 5:

Western blotting results of apoptosis and autophagy. (A) Western blot images of cleaved PARP and β-actin levels of casticin-treated KG1a and KG1 cell lines and their non-treated control groups, respectively. (B) Comparison of the cleaved PARP/β-actin optic density ratio (mean ± SEM) with corresponding control groups (** p<0.01). (C) Western blot images of LC3B-I, LC3B-II, and β-actin protein levels of casticin-treated KG1a and KG1 cell lines and their non-treated control groups. (D) Comparison of the LC3B I/LC3B II optic density ratio (mean ± SEM) with corresponding control groups. (E) Western blot images of SQSTM (p62) and β-actin levels of casticin-treated KG1a and KG1 cell lines and their non-treated control groups. (F) Comparison of the SQSTM/β-actin optic density ratio (mean ± SEM) with corresponding control groups.

The involvement of autophagy in casticin-mediated cell death was investigated by detecting the expression of autophagic proteins LC3B I/II and SQSTM. However, the results showed that casticin treatment did not significantly alter the ratio of LC3BI/II and SQSTM/β-actin in KG1a and KG1 cell lines compared to the control groups (p>0.05) (Figure 5C–F).

Western blotting analyses of the most interactive proteins (Shh, Gli and Wnt) showed that 2 μM casticin treatment caused a decrease in Gli/β-actin ratio in KG1a, KG1 cell lines after 2 μM casticin treatment compared to their control groups (KG1a and KG1 p<0.01) (Figure 6A and B). When we assessed the transcriptor factor of the Hedgehog pathway, we also found a decrease in Shh/β-actin ratio in KG1a and KG1 cell lines compared to their control groups (KG1a p<0.05; KG1 p<0.01) (Figure 6C and D). However, different results were obtained in the two cell lines for Wnt protein expressions. Following casticin treatment a decrease in Wnt protein expression was found for KG1a cells (p<0.05), but no significant changes was found in KG1cells (p>0.05) (Figure 6E and F).

Figure 6:

Western blotting results of signaling pathways. (A) Western blot images of gli and β-actin levels of casticin-treated KG1a and KG1 cell lines and their non-treated control groups. (B) Comparison of the gli/β-actin optic density ratio (mean ± SEM) with corresponding control groups (**:p<0.01; ***:p<0.001). (C) Western blot images of Shh and β-actin levels of casticin-treated KG1a and KG1 cell lines and their non-treated control groups. (D) Comparison of the Shh/β-actin optic density ratio (mean ± SEM) with corresponding control groups (*:p<0.05; **:p<0.01). (E) Western blot images of Wnt and β-actin levels of casticin-treated KG1a and KG1 cell lines and their non-treated control groups. (F) Comparison of the Wnt/β-actin optic density ratio (mean ± SEM) with corresponding control groups (**:p<0.01).

Discussion

In our present study, we investigated the underlying molecular mechanism of casticin-induced cell death by focusing on signaling pathways in in vitro LSC-like cell and AML blast models at both gene and protein expression levels.

The first aspect investigated in this study was the suitability of the in vitro models. A major feature of the LSCs is to re-initiate AML and a study found that CD34+CD38− sub-populations were consistently successful in engrafting in in vivo models of AML [31]. Therefore, to confirm our cell lines as an in vitro stem cell-like and blast model, we evaluated the CD34+ CD38- expression features of KG1a and KG1 cell lines. Our analyses of revealed that approximately 85 % of KG1a cells have the CD34+, CD38-phenotype, while 94 % of KG1 cells have the CD34+, CD38+ phenotype. In addition, it is known that KG1 cell line is the parental cell line of KG1a and unlike KG1, KG1a cell line is insensitive to the differentiation [24], [25], [26], [27], [28], [29], [30], [31], [32]. Furthermore, the French-American-British (FAB) classification system, which classifies cells according to their differentiation degree, categorizes KG1a and KG1 cell lines as M0 and M2, respectively [33]. In the light of the literature and our results, we used KG1a cell line as an in vitro stem cell-like model. Although KG1 cell line has also been used as a stem cell model in some studies [34], 35], based on our result, we used KG1 cell line as AML blast model to evaluate the effects of casticin on AML cells with different degrees of differentiation.

Aldehyde dehydrogenase 1A1 (ALDH1A1) is the predominant isoform of ALDH in mammals, protecting both hematopoietic stem cells and leukemia stem cells against reactive aldehyde metabolites [36]. ALDH1A1 is accepted as a metabolic marker of leukemia stem cells [37] and CD34+CD38− leukemic stem cells isolated from AML patients show high ALDH activity [38]. In our study, we found a significant increase in ALDH1A1 mRNA expression LSC-like KG1a cells following casticin treatment. We think that the increase in ALDH1A1 gene expression in KG1a cells may be a response to casticin-mediated anti-cancer effect. However, to fully understand the role of ALDH1A1 in casticin-mediated anti-leukemic effect, further research is needed.

Furthermore, we confirmed apoptosis-triggering effect of casticin in AML LSC-like cells and blasts through increased levels of cleaved PARP protein and flow cytometry analysis. We also assessed the role of autophagy in casticin’s anti-leukemic effect. Although many studies reported the anti-leukemic effects of some polyphenols occurred via induction of autophagy [39], [40], [41], casticin treatment did not induced autophagic flux, evaluated by assessing LC3B II/LC3B I and SQSTM (p62) levels in KG1a and KG1 cell lines. Therefore, we speculate that casticin may not induce autophagy in LSC-like cells and blasts in AML, but it has the capacity to trigger apoptosis in both cells.

Studies have shown that maintenance of LSC survival and drug resistance are ensured by survival signaling pathways, particularly the Hedgehog and Wnt-β-catenin pathways in AML [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43]. Shh, a Hedgehog pathway ligand, binds to the transmembrane patched (PTC) receptor, which regulates the translocation of Smoothened (SMO) into the cilium to alter the function of Gli transcription factors for survival effect [44]. Studies reported that GLI1 gene is overexpressed in relapsed or drug-resistant AML patients [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45]. Zhang et al. reported that casticin targets the Hedgehog signaling pathway and reduces key epithelial-mesenchymal transition factors, potentially decreasing cancer cell migration in the ovarian cancer SKOV3 cell line [46]. In our study, casticin treatment led to decrease in SHH and GLI mRNA expression, which was also confirmed at protein level. To suppress hedgehog pathway, Glasdegib, a SMO inhibitor, has been used in AML treatment for several years, but research showed that inhibition of SMO is not sufficient to trigger cell death due to the major survival effect of Gli, especially in LSCs [47], [48], [49], [50]. Therefore, we think that casticin-mediated apoptosis through inhibition of Shh ligand and Gli transcription factor in LSC-like cells and blasts might be a promising outcome for drug development.

In addition to Shh pathway, studies also showed that Wnt/β-catenin pathway also supports LSC maintenance directly or through interaction with oncogenic pathways such as Hedgehog [14], 15] and inhibition of Wnt leads to LSC death [51], 52]. In parallel with the literature, we detected decreased WNT mRNA expression after casticin treatment in the cell lines. However, in protein level, the decrease of Wnt was only confirmed for KG1a cells. Therefore, we speculated that it might be related to higher dependence of LSCs on Wnt/β-catenin signaling for survival. Moreover, Su et al. reported that Shh activation promotes cell survival via interaction with Wnt/β-catenin signaling in CD34+ chronic myeloid leukemia cells [53] and our pathway interaction analysis revealed that there is a possible crosstalk between Hedgehog signaling and Wnt. Hence, further studies are needed to uncover possible crosstalk between Hedgehog signaling and Wnt in casticin-induced cell death in AML.

In this study, we aimed to understand the effects of casticin on AML LSC-like cell line and its mature form. However, we believe that further studies, including an in vivo AML model that enables us to consider AML microenvironment and drug metabolism, might overcome the limitations of the study. Moreover, our bioinformatics analyses indicated a possible crosstalk between Hedgehog and the proteins of the MAPK, PI3K/Akt/mTOR, Notch, Hippo signaling cascades after treatment with casticin. Therefore, we think that investigating oncogenic pathway interactions may enhance long-term treatment efficacy in AML, but further studies are necessary to explore these interactions.

In conclusion, our study demonstrated for the first time that casticin induces apoptosis in AML LSC-like cells and blasts primarily by suppressing the Hedgehog signaling pathway (Figure 7). Based on these results, we propose that casticin holds a potential as a candidate for AML drug development.

Figure 7:

The possible effect of casticin on LSCs and leukemic cells.

Corresponding author: Semra Koçtürk, Faculty of Medicine, Department of Biochemistry, Dokuz Eylül University, Izmir, Türkiye, E-mail: semra.kocturk63@gmail.com

Funding source: Dokuz EylÃ¼l University, Department of Scientific Research Project

Award Identifier / Grant number: BAP 2020.KB.SAG. 035

Research ethics: The local Institutional Review Board deemed the study exempt from review.
Informed consent: Not applicable.
Author contributions: concept – S.K., T.E.; design – S.K., K.Y.A., T.E., G.G.; supervision – S.K., K.Y.A.; resources – S.K., K.Y.A., H.A.; materials – S.K., T.E., H.A.; data collection and/or processing – T.E., K.Y.A., G.G., H.A.; analysis and/or interpretation – T.E., S.K., K.Y.A., G.G., H.A.; literature search –T.E., S.K., K.Y.A., G.G.; writing –T.E., S.K., K.Y.A., G.G.; critical reviews – S.K., K.Y.A., T.E., G.G., H.A.
Use of Large Language Models, AI and Machine Learning Tools: None declared.
Conflict of interest: The authors state no conflict of interest.
Research funding: This study was funded by Dokuz Eylül University, Department of Scientific Research Project (Project no: BAP 2020.KB.SAG. 035).
Data availability: The data supporting the findings of this study can be accessed by contacting the corresponding author, S.K., upon a reasonable request.

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Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/tjb-2024-0149).

Received: 2024-06-25

Accepted: 2024-10-10

Published Online: 2024-12-03

This work is licensed under the Creative Commons Attribution 4.0 International License.

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https://doi.org/10.1515/tjb-2024-0149

Keywords for this article

acute myeloid leukemia; apoptosis; cancer stem cells; casticin; cell signaling

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