Piper nigrum Has an Antitumor Effect Through Regulation of the Phosphatidylinositol 3‐Kinase Signaling Pathway in a Murine Model of Acute Myeloid Leukemia

ABSTRACT

The search for safer and more effective therapies for acute myeloid leukemia (AML) has intensified due to low response rates and resistance to current chemotherapy. The aim of this study was to evaluate the antitumor and immunomodulatory effects of Piper nigrum extract in a murine model of AML, with particular emphasis on its ability to modulate the phosphatidylinositol 3‐kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) signaling pathway. The chemical composition of the extract was characterized using advanced analytical techniques. In vitro assays were performed to evaluate cytotoxic and cytostatic effects, glucose consumption and ROS production, modulation of Gal‐9 (galectin 9) and PD‐L1 (programmed death‐ligand 1), focusing on and relating it to the modulation of the (PI3K/AKT/mTOR) pathway. In vivo studies were performed using mice inoculated with DA‐3/ER‐GM cells to evaluate hematological parameters, survival, and immune response after P. nigrum treatment. P. nigrum extract demonstrated cytotoxic and cytostatic activity in vitro, characterized by a decrease in cell proliferation and survival possibly associated with modulation of the PI3K/AKT/mTOR pathway. In the murine AML model, treatment significantly reduced circulating blasts, improved hematological parameters, and prolonged survival. In addition, an increase in effector memory CD8⁺ T cells was observed, along with a reduction in plasma proinflammatory cytokine levels. Piper nigrum may represent a promising complementary therapeutic strategy for AML, acting as an antitumor and immunometabolic modulator. These findings suggest potential clinical applications of P. nigrum extract as an adjuvant or alternative therapy in the treatment of AML, warranting further preclinical and clinical investigation.

Overview of the antileukemic effects of Piper nigrum extract in AML. P. nigrum reduced leukemic cell proliferation and glycolytic metabolism through modulation of the PI3K/AKT/mTOR pathway. In vivo, treatment reduced leukemic burden, improved hematological parameters, prolonged survival, and modulated antitumor immune responses. Collectively, these findings support a mechanism involving metabolic and immune modulation associated with inhibition of PI3K/AKT/mTOR signaling.

1. Introduction

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic neoplasm characterized by uncontrolled proliferation and accumulation of immature and nonfunctional myeloid cells resulting from genetic alterations in hematopoietic progenitors (Saultz and Garzon 2016). Metabolic reprogramming has emerged as a key mechanism that promotes tumorigenesis and represents a potential therapeutic target to inhibit tumor growth (Phan et al. 2014; Castro et al. 2019). AML cells predominantly rely on aerobic glycolysis, a metabolic phenotype characterized by increased glucose uptake and lactate production to sustain their bioenergetic and biosynthetic demands. This metabolic shift also contributes to immune evasion and disease progression (Rashkovan and Ferrando 2019).

One of the mechanisms involved in maintaining this glycolytic phenotype is the activation of the phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, which regulates the transcription of several metabolic and survival‐related proteins, including glucose transporters (GLUT), hexokinase‐2 (HK‐2), glucose‐6‐phosphate dehydrogenase (G6PD), hypoxia‐inducible factor‐1α (HIF‐1α), and the MYC oncogene (Fontana et al. 2024). In addition to its metabolic effects, this pathway has been implicated in the regulation of immunosuppressive molecules such as galectin‐9 (Gal‐9) and programmed death ligand‐1 (PD‐L1) (Taghiloo et al. 2022; Quan et al. 2022).

Gal‐9 plays an important role in immune regulation by binding to receptors on T cells and other immune cells, inducing apoptosis or functional exhaustion. Similarly, PD‐L1 interacts with the PD‐1 receptor on T cells, delivering inhibitory signals that reduce T‐cell effector activity. Together, these mechanisms contribute to immune evasion and support the survival and progression of leukemic cells (Cao et al. 2023). Therefore, activation of the PI3K/AKT/mTOR pathway is considered a hallmark of leukemogenesis and has been associated with poor prognosis in AML patients. Constitutive activation of this signaling pathway has been reported in a significant proportion of AML cases and is linked to increased leukemic cell survival, metabolic reprogramming, and resistance to conventional therapies (Liu et al. 2022; Nepstad et al. 2020). Although several pharmacological inhibitors targeting this pathway have been developed, their clinical efficacy in AML has been limited due to toxicity, incomplete pathway inhibition, and the emergence of resistance mechanisms (Huang et al. 2021). Consequently, the identification of alternative or complementary therapeutic strategies capable of modulating this pathway remains an important challenge.

In this context, natural products have gained increasing attention as potential modulators of oncogenic signaling pathways due to their multitarget properties and relatively favorable safety profiles. Compounds derived from Piper nigrum , commonly known as black pepper, have shown promising biological activities. Piperine and related alkaloids present in the outer layer of the fruit have been reported to exert inhibitory effects on the PI3K/AKT/mTOR signaling pathway (Hakeem et al. 2024; Han et al. 2023). These compounds exhibit several antitumor properties, including inhibition of metalloproteinases, modulation of epithelial–mesenchymal transition, suppression of angiogenesis, and regulation of apoptotic proteins such as Bax and Bcl‐2 (Yoo et al. 2019; Mitra et al. 2022).

Previous work from our group demonstrated that an ethanolic extract of P. nigrum significantly reduced tumor size and decreased macrometastasis in the 4T1 breast cancer and B16‐F10 melanoma models. In addition, treatment with P. nigrum extract increased the frequency of dendritic cells and activated CD8⁺ T cells while reducing the frequency of myeloid‐derived suppressor‐like cells (MDSC‐LCs) and regulatory T cells (Tregs) within the tumor microenvironment (Lasso et al. 2023). These findings suggest that P. nigrum extract may exert both antitumor and immunomodulatory effects.

To evaluate these effects in AML, we employed the DA‐3/ER‐GM murine leukemia cell line, a granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐producing leukemia model derived from the DA‐3 murine myeloid leukemia line. DA‐3/ER‐GM cells display myeloid lineage characteristics, rapid proliferation, and reproducible induction of aggressive leukemia when inoculated into syngeneic BALB/c mice. Due to these features, this model has been widely used to study leukemia progression, tumor‐induced immunosuppression, and the evaluation of novel therapeutic strategies in vivo.

Therefore, the aim of this study was to evaluate the antitumor and immunomodulatory effects of P. nigrum extract in a murine model of AML, with particular emphasis on its ability to modulate the PI3K/AKT/mTOR signaling pathway.

2. Materials and Methods

2.1. Plant Material

The fruits of the P. nigrum plant were harvested during the vegetative stage in Orito, Putumayo, Colombia (0°37′45″ N, 76°51′55″ W). The taxonomic characterization of the species was carried out by the biologist Néstor García at the herbarium of the Pontificia Universidad Javeriana (specimen number 30548). Eighty grams of the plant material was taken for extraction by percolation with 96% ethanol and concentrated under reduced pressure by rotary evaporation as previously described. The finally dried extract was stored at room temperature for further uses. The chemical characterization of the extract was performed as described previously (Lasso et al. 2023; Bravo‐Chaucanés et al. 2022).

2.2. Tumor Cell Lines and Culture Conditions

The DA‐3/ER‐GM murine leukemia cell line, a granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐producing AML model originally derived from the DA‐3 murine myeloid leukemia line. DA‐3/ER‐GM cells were provided by Professor José Arteaga (Department of Basic Sciences, Faculty of Medicine, Universidad Industrial de Santander, Bucaramanga, Colombia); and were maintained in RPMI‐1640 medium (Eurobio, Toulouse, France) supplemented with 10% heat‐inactivated fetal bovine serum (FBS), 2 mM l‐glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 0.01 M HEPES buffer, 1 mM sodium pyruvate (Eurobio), and 50 μg/mL gentamicin (Gibco, Grand Island, NY, USA) and were cultured at 37°C in 5% CO₂.

The human leukemic cell lines K562 (chronic myelogenous leukemia, isolated from bone marrow; ATCC CCL‐243) and U937 (histiocytic lymphoma, pro‐monocytic cells isolated from pleural effusion; ATCC CRL‐1593.2) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI‐1640 medium supplemented with 10% heat‐inactivated fetal bovine serum (FBS; Eurobio, Toulouse, France), 2 mM l‐glutamine, 100 U/mL penicillin–streptomycin (Gibco), 0.01 M HEPES buffer (Gibco), and 1 mM sodium pyruvate (Gibco, Waltham, MA, USA).

All cell lines were initially cultured at a concentration of 2 × 10⁵ cells/mL under standard cell culture conditions in a humidified incubator at 37°C with 5% CO₂. For all experiments, the cells were thawed from cryopreserved vials and maintained in culture for no more than two passages prior to use in the experiment. No long‐term continuous culture was performed. Culture medium was replaced every 48 h when cells reached approximately 80% confluence to ensure viability and adequate growth.

2.3. Animals

Young female BALB/cAnNCrl mice (6–10 weeks of age), weighing between 16 and 22 g, were originally obtained from Charles River Laboratories and used to establish a breeding colony at the animal facility of the Pontificia Universidad Javeriana (PUJ, Bogotá, Colombia). Animals were housed and handled in accordance with the protocols approved by the Ethics Committee of the Faculty of Sciences and in compliance with national and international regulations for experimentation with live animals (Republic of Colombia, Resolution 08430 of 1993; National Academy of Sciences 2010). All experimental procedures were approved by the Institutional Animal care and use committee (AIUC) of Pontificia Universidad Javeriana (FUA‐104‐21). Mice were maintained in polyethylene cages with ad libitum access to food and water, under a 12 h light/dark cycle at 20°C–22°C and 40%–60% relative humidity. To minimize selection bias, mice from the established colony were randomly allocated to experimental groups in equal numbers using a random assignment procedure after confirmation of tumor cell engraftment. Investigators involved in treatment administration, animal monitoring, data acquisition, and outcome assessment were blinded to group allocation. Data analysis was performed without knowledge of treatment assignment to ensure unbiased interpretation of the results.

No anesthesia or analgesia was administered during the experimental procedures, as these interventions may alter immune parameters relevant to the study. Animal welfare was closely monitored throughout the experiment by trained personnel through daily assessment of behavior, body weight, and clinical signs. Humane endpoint criteria were applied, and animals exhibiting signs of pain, distress, or loss of quality of life were euthanized immediately. All treatments were administered intraperitoneally twice per week until the end of the experimental period, at the doses described in the corresponding sections. Euthanasia was performed by carbon dioxide (CO₂) inhalation at a concentration of 5%, in accordance with institutional and international animal welfare guidelines.

2.4. In Vitro Cytotoxicity Tests

The cytotoxic effect of P. nigrum extract on DA‐3/ER‐GM tumor cells was evaluated using the methylthiazol tetrazolium (MTT) assay (Sigma–Aldrich, Saint Louis, MO, USA). DA3/ER‐GM cells (6 × 10³ cells/well) were seeded in 96‐well plates in a final volume of 200 μL of supplemented RPMI medium and treated with serial dilutions of P. nigrum extract starting at 250 μg/mL for 48 at 37°C in 5% CO₂. The IC₅₀ (50% inhibitory concentration) was calculated using the GraphPad Prism statistical software version 10.3.1 for Mac OS X (GraphPad Software, San Diego, CA, USA).

2.5. Proliferation Assay

1 × 10⁵ DA‐3/ER‐GM cells were seeded in 12‐well plates with the IC₅₀/2 of P. nigrum extract and ethanol (negative control) for 24, 48, and 72 h. Once the evaluation times were completed, the cell count for each treatment condition was performed using trypan blue. To calculate the doubling time, the formula doubling time = duration × Ln(2)/ln(final cells/initial cells) was used and the exponential growth formula (Malthusian) Y = Y ₀ × exp(k × x) of GraphPad Prism software version 10.3.1 for MacOS X was used for graphing.

2.6. OCR and ECAR Evaluation

Agilent MitoXpress and pH‐Xtra Kits were used to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) on the Cytation5 reader (BioTek, Winooski, VT, USA), following the instructions. ECAR and OCR were measured simultaneously under basal conditions in three leukemic cell lines: DA‐3/ER‐GM, U937 and K562. Cells were seeded in a 96‐well plate at a density of 1.25 × 10⁴ per well. Before measurement, controls were added: 50 mM 2‐DG (2‐deoxy‐d‐glucose > 98%, Sigma–Aldrich, Burlington, MA, USA) was used for ECAR, and for OCR, 1 μM AntiA (Streptomyces sp. Antimycin A, Sigma‐Aldrich, St. Louis, MO, USA) was used. TR‐F reading and lifetime calculation determined the fluorescence depletion rate of the MitoXpress Xtra and pH‐Xtra reagents. The dual intensity readings were used to calculate the corresponding life time (μs) by the following equation: life time (μs)[τ] = (D ₂ − D ₁)/ln(IW ₁/IW ₂), where IW ₁ and IW ₂ represent the two (dual) measurement points and D ₁ and D ₂ represent the delay time before measurement of W ₁ and W ₂, respectively.

2.7. Glucose Uptake Assay

To assess glucose uptake under basal conditions, cells from human (K562 and U937) and murine (DA‐3/ER‐GM) leukemic cell lines were used and compared with femoral medullary aspirate and peripheral blood cells from healthy young BALB/cAnNCrl mice. To determine the effect of P. nigrum on DA‐3/ER‐GM cells, 3 × 10⁵ cells were treated in 12‐well plates with the IC₅₀ of P. nigrum extract and 25 μM of the kinase inhibitor control LY294002 (Life Technologies, Thermo Scientific, Eugene, OR, USA) for 6, 12, and 24 h. Cells were washed with 1× PBS prior to labeling with 40 μM of 2‐NBDG (2‐(N‐(7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)Amino)‐2‐Deoxyglucose) (Invitrogen Molecular Probes) prepared in RPMI 1640 without phenol red. Cells were incubated for 30 min at 37°C with the probe and washed with cold 1× PBS. The distinction of live versus dead cells was performed by labeling with LIVE/DEAD Fixable Aqua (Life Technologies, Thermo Scientific, Eugene, OR, USA). Finally, samples were acquired on a Cytek Aurora Spectral Cytometer (Cytek Biosciences, Fremont, CA, USA) and analyzed with FlowJo v 10.10.0 software (BD Life Sciences, Franklin Lakes, NJ, USA). Experiments were performed in triplicate, and results were expressed as mean ± standard error of the mean (SEM).

2.8. Measurement of Reactive Oxygen Species (ROS)

To assess ROS production, 1.5 × 10⁵ leukemic cell lines were treated in 12‐well plates with the IC₅₀ of P. nigrum extract or 0.02 μM of Doxorubicin (prooxidant control) (MP Biomedicals, Santa Ana, CA, USA) for 12 and 24 h. Then, cells were count and stained with 1 μM H2DCFDA (2′,7′‐dichlorodihydrofluorescein diacetate) (Sigma Aldrich, Saint Louis MO, USA) for 40 min at 37°C, followed by propidium iodide (PI) (Sigma‐Aldrich) used as a viability dye. Samples were acquired on the Cytek Aurora Spectral Cytometer (Cytek Biosciences, Fremont, CA, USA) and analyzed with FlowJo v10.10.0 software (BD Life Sciences, Franklin Lakes, NJ, USA). Experiments were performed in triplicate, and results were expressed as mean ± SEM.

2.9. Evaluation of Intracellular Phosphorylated Proteins by Flow Cytometry

3 × 10⁵ DA‐3/ER‐GM cells were first stained with the viability stain LIVE/DEAD Fixable Aqua (Life Technologies) for 20 min in the dark at room temperature. Cells were fixed and permeabilized using the BD Cytofix/Cytoperm Fixation/Permeabilization Solution kit (BD Bioscience) and then stained for 30 min at room temperature with intracellular specifics antibodies for the phosphorylated proteins p‐PI3K, p‐AKT1, and p‐mTOR. To assess the phosphorylation of these proteins under basal conditions, we used T cells from the spleen of BALB/cAnNCrl mice. Activation was performed with anti‐CD3 (clone 145‐2C11) and anti‐CD28 (clone 37.51) (BD Bioscience) for 24 h, and unstimulated cells were used as a negative control. For tumor cells, activation was performed by culturing the cells with GM‐CSF (20 ng/mL) (R&D Systems) for 24 h. As a negative control, cells were treated with the inhibitor LY294002 (25 μM) for 24 h. To evaluate the effect of P. nigrum extract on this pathway, DA‐3/ER‐GM cells were treated with the IC₅₀ concentration for 24 h. The obtained data were analyzed using FlowJo software v10.10.0 (BD Life Sciences, Franklin Lakes, NJ, USA), and all experiments were performed in triplicate, with results expressed as mean ± SEM (Table S1). The original flow cytometry plots showing the variation in metabolic subpopulations (Hi, Int, and Low), as well as the changes in the frequencies of PI3K/AKT/mTOR pathway activation in cells under the evaluated treatments, are provided in the Supporting Information. Additionally, summary tables containing these data and the corresponding fold change calculations are included in Supporting Information (Figures S2 and S3 and Tables S2 and S3).

2.10. Evaluation of Immunosuppressive Molecules

DA‐3/ER‐GM cells (3 × 10⁵) were treated for 24 h with the IC₅₀ of P. nigrum extract, LY294002 (25 μM, negative control), IFN γ (10 ng/mL, positive control) (BioLegend, San Diego, California), IFN γ plus P. nigrum extract, and IFN γ plus LY294002. After the treatments, cells were washed with 1× PBS and stained with LIVE/DEAD Fixable Aqua for 20 min in the dark at room temperature. After washing with 2% FBS‐PBS, the cells were stained for 30 min at 4°C with anti‐PD‐L1. Subsequently, the cells were fixed and permeabilized with the Cytofix/Cytoperm Kit followed by staining with anti‐Gal‐9 and then with the secondary antibody Alexa Fluor‐488 goat anti‐rabbit. PD‐L1 and Gal‐9 expression levels were quantified by flow cytometry as the median fluorescence intensity (MFI). For data analysis, results were expressed as fold change relative to untreated control cells. In each independent experiment, the MFI value of untreated cells was normalized to 1, and the MFI values of all treatment conditions were calculated relative to this baseline.

This normalization strategy enabled comparison across independent experiments and minimized variability associated with cytometer acquisition settings, staining efficiency, or baseline fluorescence between assays. Accordingly, the dotted horizontal line shown in the graphs represents the basal expression level of untreated cells (negative control).

Experiments were performed in three independent biological replicates, each conducted in triplicate. In this context, values greater than 1 indicate increased expression relative to untreated cells, whereas values below 1 indicate decreased expression compared with basal levels. Samples were acquired using a Cytek Aurora cytometer (Cytek Biosciences, Fremont, CA, USA), and data were analyzed with FlowJo software v10.10.0 (BD Life Sciences, Franklin Lakes, NJ, USA). Results are presented as mean ± SEM. Additional experimental details are provided in Table S1.

2.11. In Vivo AML Model and Therapeutic Potential of P. nigrum Extract

The in vivo model was performed by inoculating 5 × 10⁴ DA‐3/ER‐GM cells into the lateral tail vein of young BALB/cAnNCrl mice. To evaluate the antitumor effect of different treatments on the development of leukemogenesis, 3 days after tumor cell inoculation, mice were treated twice per week by intraperitoneal injection with P. nigrum extract (15.6 mg/kg), LY294002 (25 mg/kg), doxorubicin (1 mg/kg), or PBS as vehicle control. The injection volume was adjusted according to body weight and did not exceed 200 μL per administration. The dose of P. nigrum extract was selected based on previous acute toxicity studies in BALB/c mice, corresponding to approximately fourfold lower than the calculated LD₅₀. Treatments were continued until animals reached predefined humane endpoint criteria associated with leukemia progression (Figure 5A).

FIGURE 5.

Piper nigrum extract exerts an antitumor effect and prolongs survival in a murine model of acute myeloid leukemia. (A) Experimental design to evaluate the antitumor effect of P. nigrum in AML model with DA‐3/ER‐GM cells. (B) Percentage of circulating blasts assessed with a wright‐stained peripheral blood smear. (C) Percent neutrophils in peripheral blood. (D) Percent lymphocytes in peripheral blood. (E) Percentage survival during the disease in PBS‐treated (untreated) animals (gray line), doxorubicin‐treated animals (red line), and P. nigrum ‐treated animals (blue line). Hematological parameters shown in panels (C) and (D) were measured from peripheral blood obtained by cardiac puncture immediately after euthanasia. Due to coagulation or insufficient sample recovery in some animals, only five animals per group (n = 5) were included in these analyses. Data shown in panels (B) and (E) correspond to all animals analyzed (n = 8 per group). p values indicate statistically significant differences between treatments. p < 0.05; p < 0.01; p < 0.001; p < 0.0001.

Mice were monitored three times per week, evaluating body weight, well‐being and hematological parameters using the Mindray BC‐5150 (Mindray Bio‐Medical Electronics Co. Ltd., Shenzhen, China) and a peripheral blood smear stained with Wright's technique using an automatic dye (Aerospray Stainer, Wescor, Logan, UT, USA). Animals were sacrificed by CO₂ inhalation when endpoint criteria were reached. Immediately, blood samples were collected by cardiac puncture, and then spleen, femurs and lymph nodes were obtained to assess metastasis by the presence of blasts in these organs.

2.12. Quantification of Cytokine Levels in Serum

Plasma samples were collected via cardiac puncture upon euthanasia of in vivo AML model mice and stored at −80°C prior to analysis. Serum cytokine levels were determined according to the manufacturer's instructions using a Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 kit (BD Biosciences). This assay, based on multiplex flow cytometry, employs different populations of fluorescent capture beads coated with cytokine‐specific antibodies so that multiple cytokines can be detected at one time in a single sample. In brief, 50 μL of plasma was incubated for 2 h at room temperature in the dark with 50 μL mixed cytokine capture beads and 50 μL of PE‐conjugated detection reagent. Following the incubation, beads were washed with wash buffer and centrifuged, then resuspended in 300 μL of wash buffer for acquisition. Samples were obtained using a FACSAria II flow cytometer (BD Immunocytometry Systems). Individual bead populations were discriminated according to their intrinsic fluorescence intensity, and cytokine concentrations were derived from the phycoerythrin (PE) fluorescence signal deriving from each bead population. Standard curves were generated from the cytokine standards that accompanied the kit, and concentrations of cytokine were calculated using FCAP Array software version 3.0 (BD Biosciences).

2.13. Evaluation of Immune Populations

To evaluate the functional activity of T cells after treatment with P. nigrum extract, LY294002, and Doxorubicin (positive control), 1 × 10⁶ splenocytes were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain for 20 min in the dark at room temperature, then washed with 2% FBS‐PBS, and stained for 30 min at 4°C in the dark with anti‐CD3, anti‐CD4, anti‐CD8, anti‐CD25, anti‐CD44, anti‐CD62L, anti‐CD69, anti‐TIM‐3, and anti‐ PD1 (Table S1). Subsequently, cells were washed, fixed and permeabilized with True‐ Nuclear transcription Factor Buffer set and stained with anti‐CTLA‐4 and anti‐FOXP3 for 45 min at room temperature in the dark. Finally, cells were washed and resuspended in 300 μL of 1× PBS and acquired by flow cytometry using Cytek Northern Lights‐CLC cytometer (Cytek Biosciences, Fremont, CA, USA), and the results were subsequently analyzed using FlowJo 10.10.0 software.

2.14. Best Practice in Natural Products Pharmacological Research

The study design, experimental procedures, and reporting of results were conducted in accordance with recommendations for best practices in pharmacological research involving natural products, including considerations for extract characterization, experimental design, and data interpretation, as outlined in recent guidelines (11, 12).

2.15. Statistical Analysis

All statistical analyses were performed using GraphPad Prism version 10.3.1 for Mac OS X (GraphPad Software, San Diego, CA, USA). Data are presented as mean ± SEM.

Prior to statistical testing, data distribution was assessed using the Shapiro–Wilk normality test. As several datasets did not meet the assumptions of normal distribution and given the sample size, non‐parametric tests were applied throughout the study.

Comparisons between two independent groups were performed using the Mann–Whitney U test. Comparisons among multiple groups were conducted using the Kruskal–Wallis test followed by Dunn's multiple comparisons post hoc test when appropriate.

Data were not normalized or transformed prior to analysis. A p value < 0.05 was considered statistically significant.

3. Results

3.1. Piper nigrum Has a Cytotoxic and Cytostatic Activity on the Highly Metabolic DA‐3/ER‐GM Cells

To evaluate the cytotoxic activity of P. nigrum extract, DA‐3/ER‐GM cells were treated with different concentrations of the extract for 48 h. A dose‐dependent cytotoxic activity was observed, with an IC₅₀ value of 13 ± 3 μg/mL (Figure 1A). Additionally, a cytostatic activity at a nonlethal concentration (IC₅₀/2) was observed, evidenced by an increase in cell doubling time to 17 h compared to 12 h in ethanol‐treated control cells. Importantly, ethanol used as vehicle control did not induce any cytotoxic effect, maintaining cell viability above 98% at the highest evaluated concentration (250 μg/mL) (Figure 1B; Figure S1).

FIGURE 1.

Cytotoxic and cytostatic effects of Piper nigrum extract on DA‐3/ER‐GM cells. (A) Dose–response viability curve of DA‐3/ER‐GM cell line treated with P. nigrum extract or ethanol (vehicle). (B) DA‐3/ER‐GM cell count per cm² after treatment with P. nigrum extract or ethanol (vehicle). Population doubling times (PDT) are shown for each treatment. Data are presented from three independent experiments performed in duplicate for both cytotoxicity and proliferation assays. p values indicate statistically significant differences between treatments. *p < 0.05.

The metabolic profile of DA‐3/ER‐GM cells was evaluated by measuring the Extracellular Acidification Rate (ECAR) and the OCR. DA‐3/ER‐GM cells exhibited high glycolytic activity (ECAR: 51), like the glycolytic cell lines K562 (ECAR: 53.7) and U937 (ECAR: 52.7). Additionally, mitochondrial oxidative phosphorylation was active in DA‐3/ER‐GM cells, with an OCR value of 7.23, comparable to K562 cells (OCR: 7.3) but significantly higher than U937 cells (OCR: 1.6). These results indicate that DA‐3/ER‐GM cells have a flexible metabolic profile, effectively utilizing both glycolysis and oxidative phosphorylation to fulfill their energetic requirements. Such metabolic flexibility resembles that previously reported for the K562 cell line, in contrast to the predominantly glycolytic and less oxidative profile observed in U937 cells (Figure 2A).

FIGURE 2.

Metabolic activity in DA‐3/ER GM cells: Increased glucose consumption, reactive oxygen species (ROS) production, and activation of the PI3K/AKT/mTOR pathway. (A) Energy map comparing extracellular acidification rate (ECAR, mpH/min) and oxygen consumption rate (OCR, pmol O₂/min) in leukemic cell lines (K562, U937, and DA‐3/ER‐GM) under basal conditions. The analysis reveals that DA‐3/ER‐GM cells exhibit a high glycolytic activity comparable to that observed in K562 and U937 cells, while maintaining active oxidative phosphorylation. This metabolic profile indicates that DA‐3/ER‐GM cells display metabolic flexibility, enabling them to utilize both glycolysis and mitochondrial respiration to sustain their energetic demands. Data were obtained from two independent experiments performed in duplicate. (B) Mean fluorescence intensity (MFI) of 2‐NBDG (2‐Deoxy‐2‐[(7‐nitro‐2, 3‐benzoxadiazol‐4‐yl) amino]‐d‐glucose) in DA‐3/ER‐GM, K562, U937 cells and murine peripheral blood and bone marrow hematopoietic cells under basal conditions. Data were obtained from three independent experiments performed by triplicate. (C) H₂DCFDA (diacetate de 2′,7′‐diclorofluorescein) MFI values in DA‐3/ER‐GM, K562, and U937 cells. Data were obtained from two independent experiments performed in duplicate. (D) Mean fluorescence intensity (MFI) of phosphorylated proteins from the PI3K/AKT/mTOR pathway in tumor cells compared to normal cells, evaluated under basal conditions, following stimulation with GM‐CSF or anti‐CD3 and anti‐CD28 antibodies in T cells, and after treatment with the inhibitor LY294002. Due to the limited availability of phospho‐specific antibodies (5 μL per sample), the analysis of the PI3K/AKT/mTOR pathway was experimentally prioritized in DA‐3/ER‐GM cells of interest, performing a higher number of replicates for this cell line compared to other cell populations used as controls. Measurements were performed twice using a single biological sample for T cells and the U937 cell line. In contrast, DA‐3/ER‐GM cells were analyzed in two independent experiments, each performed in duplicate. p values were calculated using the nonparametric Mann–Whitney test for comparisons between two groups.*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

On the other hand, DA‐3/ER‐GM cells, together with U937 and K562 leukemic cells show a high rate of glucose uptake compared to bone marrow and peripheral blood hematopoietic cells from a healthy mouse (Figure 2B). Furthermore, DA‐3/ER‐GM cells have elevated levels of ROS compared with the other cell lines evaluated (Figure 2C).

Then, the PI3K/AKT/mTOR signaling pathway, which has been implicated in the maintenance of glycolysis, proliferation, and survival in hematopoietic tumors was evaluated. Under basal conditions, DA‐3/ER‐GM cells exhibit constitutive activation of PI3K/AKT/mTOR phosphorylation, similar to U937 cells, known to have a constitutive activation (13). However, after stimulation with GM‐CSF, the phosphorylation of AKT (p‐AKT) and mTOR (p‐mTOR) was significantly increased in DA‐3/ER‐GM and only a few on U937 cell line. Treatment with the kinase inhibitor LY294002 significantly reduced the phosphorylation of AKT (p‐AKT) and mTOR (p‐mTOR) in DA‐3/ER‐GM cells. In contrast, normal T cells exhibited significantly lower levels of protein phosphorylation compared to tumor cells, even when activated (Figure 2D). These results suggest that DA‐3/ER GM cells exhibit increased glucose consumption and increased activation of the PI3K/AKT/mTOR pathway compared to normal hematopoietic cells but lower than U937, which may contribute to their proliferation and survival in the tumor context.

3.2. Piper nigrum Decreases the Metabolically Active Tumor Population and Modulates the PI3K/Akt/mTOR Signaling Pathway

Three cellular subpopulations—high, intermediate, and low—were identified in DA‐3/ER‐GM cells based on their SSC‐A complexity and FSC‐A size (Figure 3A). Each subpopulation was evaluated for metabolic parameters such as glucose uptake and reactive oxygen species (ROS) levels. The high‐complexity and larger subpopulation demonstrated the highest glucose consumption and ROS production, indicating elevated metabolic activity. The intermediate subpopulation displayed moderate levels of glucose uptake and ROS production, suggesting intermediate metabolic activity, whereas the low‐complexity and smaller subpopulation showed the lowest levels of these metabolic markers (Figure 3B,C). After 24 h of treatment with the P. nigrum extract, there was a noticeable reduction in the proportion of the metabolically active high‐complexity subpopulation, accompanied by an increase in both intermediate and especially low‐complexity populations compared to untreated baseline conditions. A similar but more pronounced effect was observed following treatment with the kinase inhibitor LY294002, reflected by a substantial increase in the low‐complexity population (Figure 3D). Finally, the impact of P. nigrum extract on the PI3K/AKT/mTOR signaling pathway, known to regulate metabolic complexity, was evaluated. Both P. nigrum extract and LY294002 significantly reduced the phosphorylation levels of PI3K, AKT, and mTOR compared to the basal conditions (Figure 3E).

FIGURE 3.

Piper nigrum reduces metabolically active tumor cells and modulates the PI3K/AKT/mTOR pathway. (A) Analysis strategy for the selection and identification of DA‐3/ER‐GM cell subpopulations: High (Hi), intermediate (Int), and low. (B) Histogram and MFI of 2NBDG in each subpopulation. (C) Histogram and MFI of H₂DCFDA in each subpopulation. (D) Distribution and percentage of DA‐3/ER‐GM cells untreated and treated with P. nigrum extract and LY294002 for 24 h, comparing SSC‐A and FSC‐A. (E) Fold change (FC) in the percentage of phosphorylated PI3K, AKT and mTOR in DA‐3/ER‐GM cells after treatment with P. nigrum extract or the PI3K inhibitor LY294002 for 24 h. Data are expressed relative to untreated cells (basal condition), which were normalized to a value of 1 and are represented by the dotted line. Panels (A–C) correspond to two independent experiments performed in duplicate, whereas panels (D and E) correspond to three independent experiments performed in duplicate. p values were calculated using a nonparametric Mann–Whitney test for comparisons between two groups. p < 0.05, p < 0.01, p < 0.001, p < 0.0001.

Since the PI3K/AKT/mTOR pathway is a central regulator of cellular metabolism, including glycolysis, glucose uptake, and anabolic growth, its inhibition may contribute to the observed reduction in the highly metabolically active tumor subpopulation. Therefore, the decrease in high‐complexity DA‐3/ER‐GM cells after P. nigrum treatment is consistent with the suppression of this signaling pathway, which may limit the metabolic fitness and survival of leukemia cells.

3.3. Piper nigrum Decreases Glucose Uptake, Gal‐9 Expression, and Increases Intracellular ROS in DA‐3/ER‐GM Cells

The PI3K/AKT/mTOR pathway not only regulates cellular metabolism and survival but also actively modulates immune responses by inducing immunosuppressive molecules such as PD‐L1 and Galectin‐9 (Gal‐9), thereby contributing to immune evasion in various cancers. In this context, we evaluated the effect of P. nigrum extract on cellular metabolic activity, oxidative stress, and the expression of PD‐L1 and Gal‐9, as well as its potential association with inhibition of this signaling pathway.

A kinetic analysis of glucose uptake revealed that P. nigrum extract induced a significant reduction in glucose uptake at 6 h posttreatment. However, no further reduction was observed at later time points (12 and 24 h), suggesting an early effect on glycolytic activity (Figure 4A). In contrast, the PI3K inhibitor LY294002 resulted in a sustained and significant inhibition of glucose uptake at all time points assessed (6, 12, and 24 h), consistent with its direct action on a key metabolic pathway.

FIGURE 4.

Piper nigrum decreases glucose uptake, increasing ROS production, and reducing Gal‐9 expression in DA‐3/ER‐GM cells. (A) Fold change (FC) of MFI for 2‐NBDG in DA‐3/ER‐GM cells treated with P. nigrum extract or LY294002. (B) Fold change (FC) of MFI for H₂DCFDA in DA‐3/ER‐GM cells treated with P. nigrum extract or Doxorubicin. (C) Fold change (FC) of the MFI of PD‐L1 expression after treatment with P. nigrum , LY294002, IFN γ and treatments combinations. (D) Fold change (FC) of the MFI of Gal9 expression after respective treatments. Data are expressed relative to untreated cells (basal condition), which were normalized to a value of 1 and are represented by the dotted line in each graph. Therefore, all values correspond to changes relative to the untreated control condition. Data are presented from three independent experiments performed in triplicate. p values were calculated using the Mann–Whitney U‐test. *p < 0.05, **p < 0.01.

In parallel, oxidative stress analysis showed that P. nigrum extract significantly increased the production of ROS at 12 and 24 h posttreatment, reaching levels comparable to those induced by the pro‐oxidant agent doxorubicin used as a positive control (Figure 4B). These results suggest that P. nigrum exerts a cumulative pro‐oxidant effect on tumor cells, potentially contributing to its antitumor activity.

Regarding the expression of immunosuppressive ligands, neither P. nigrum extract nor LY294002 significantly altered PD‐L1 levels under basal conditions or following IFN‐γ stimulation (Figure 4C), indicating that PD‐L1 regulation is not exclusively dependent on the PI3K/AKT/mTOR pathway in this cellular model. However, Gal‐9 expression was modestly but significantly reduced under basal conditions following treatment with P. nigrum and the PI3K inhibitor, although this effect was not maintained in the presence of IFN‐γ (Figure 4D). Interestingly, co‐treatment with LY294002 showed a trend toward counteracting the IFN‐γ‐induced upregulation of Gal‐9, suggesting that Gal‐9 expression may be partially regulated by the PI3K/AKT/mTOR pathway, yet also influenced by IFN‐γ‐independent mechanisms.

Together, these findings suggest that P. nigrum extract modulates cellular metabolism and redox status and exerts selective effects on the expression of immunoregulatory molecules, potentially through mechanisms associated with but not limited to the PI3K/AKT/mTOR signaling pathway.

3.4. Piper nigrum Has an Antileukemic Effect and Improves Survival in a Murine Model of AML

In in vitro experiments, P. nigrum extract showed a potential antitumor effect, as evidenced by a decrease in glycolytic metabolism, an increased pro‐oxidative effect on ROS, modulation of the PI3K/AKT/mTOR pathway, and a decrease in the expression of Gal‐9 (galectin‐9) in DA‐3/ER‐GM leukemia cells. Based on these findings, the efficacy of the extract was evaluated in a murine model of AML to determine whether the effects observed in vitro were reproduced in a physiological context of the disease.

In the in vivo assay, 5 × 10⁴ DA‐3/ER‐GM cells were inoculated intravenously into BALB/cAnNCrl mice. Three days after inoculation, and after confirming the presence of circulating blast cells in peripheral blood, the animals were divided into four treatment groups: one received P. nigrum extract (twice a week); another received the PI3K inhibitor LY294002 (twice a week); another received a single weekly dose of doxorubicin; and a fourth group was left untreated with PBS (control). The treatments were continued until the mice showed signs of deterioration in their general condition. At that point, survival was assessed in each group (Figure 5A).

On Day 12 post‐inoculation, mice treated with P. nigrum extract showed a significant reduction in circulating blast counts in peripheral blood compared with the PBS‐treated control group (diseased, untreated animals), even exceeding the effect observed in the doxorubicin‐treated group, which was included as a treatment control given its established use as a chemotherapeutic agent for this type of cancer (Figure 5B). Additionally, both P. nigrum and doxorubicin‐treated animals maintained circulating neutrophil counts within the physiological ranges reported for healthy BALB/c mice (Figure 5C) and exhibited peripheral lymphocyte percentages consistent with normal hematological values (Figure 5D).

It is important to note that the PBS control group corresponds to leukemic animals that did not receive any form of treatment and therefore represents a model of active, untreated disease. In this context, these animals displayed a higher burden of circulating blasts and a marked disruption of hematopoiesis, reflected by features of cytopenia.

In contrast, the reduction in leukemic burden observed in the treated groups likely contributes to improved preservation of normal hematopoiesis, preventing the displacement of functional hematopoietic lineages. Consequently, the median survival of mice treated with the extract was 35 days, significantly longer than that of the untreated control group (25 days) (Figure 5E). Taken together, these findings indicate that P. nigrum extract delays disease progression in this murine model of AML.

3.5. Piper nigrum Extract Decreases Tumor Progression and Metastasis in AML Murine Model

To further evaluate the therapeutic effect of the ethanolic extract of P. nigrum on leukemogenesis and its impact on hematological, inflammatory, and immunological parameters associated with the progression of AML, the previously established murine model was used. Unlike the previous protocol, this experiment incorporated a standardized ethical endpoint: all animals were euthanized when any experimental group showed the first clinical sign of deterioration, allowing a synchronous comparison of pathophysiological alterations among treatment conditions.

Overall, the PBS‐treated control group, corresponding to leukemic animals without therapeutic intervention, exhibited progressive hematological deterioration during disease progression, as evidenced by alterations in erythrocyte count, hematocrit, platelet levels, and an increased proportion of circulating blasts. In contrast, animals treated with P. nigrum , LY294002, or doxorubicin showed a partial preservation of hematological parameters at several time points (Figure 6A–C). This effect was not uniform across all parameters or time points; for example, hematocrit values at Day 14 were below physiological reference ranges in the experimental groups. Therefore, rather than indicating complete normalization, these findings suggest a partial attenuation of leukemia‐induced hematological alterations compared with the untreated leukemic control group.

FIGURE 6.

Antitumor activity of P. nigrum in the DA‐3/ER‐GM mouse model. (A) Red blood cell count RBC (M/μL). (B) Hematocrit percentage HCT (%). (C) Platelets PLT (K/μL). (D) Frequency of circulating blasts assessed with a wright‐stained peripheral blood smear. (E) Number of tumor cells in bone marrow, spleen, lung, and lymph nodes. In all cases, eight samples per group were analyzed. Data are represented as the mean ± SEM. p values were calculated using the Mann–Whitney U‐test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Regarding circulating blasts, a statistically significant reduction was observed on Days 10 and 17 in the P. nigrum ‐treated group compared with untreated leukemic controls (Figure 6D). No statistically significant reduction was observed at Day 3 for P. nigrum ; however, at this early time point, a significant decrease was detected only in the LY294002‐treated group, which served as a positive pharmacological control due to its well‐established inhibitory effect on the PI3K/AKT/mTOR pathway. Notably, LY294002 also showed reductions in blast proportions at later time points, supporting the involvement of this pathway in leukemic progression. Doxorubicin also exhibited decreases in circulating blasts at certain time points, although its effect appeared less consistent compared with P. nigrum and LY294002.

Complementarily, postmortem analysis revealed reduced blast cell infiltration in hematopoietic organs, particularly in bone marrow and spleen, in treated animals compared with untreated leukemic controls (Figure 6E). This reduction was more evident in the P. nigrum and LY294002 groups, while doxorubicin showed a tendency toward decreased infiltration, especially in the spleen. In lymph nodes, differences among groups were less pronounced, suggesting that treatment effects on tissue dissemination may vary depending on the hematopoietic or lymphoid compartment evaluated.

Taken together, these findings indicate that P. nigrum extract exerts a relevant antileukemic effect by reducing circulating tumor burden and limiting infiltration into key hematopoietic organs. The similar pattern observed with LY294002 further supports the involvement of the PI3K/AKT/mTOR pathway in this response, while the doxorubicin group provides a clinically relevant therapeutic comparator.

3.6. Proinflammatory Cytokines Are Reduced by P. nigrum Extract

Cytokines are signaling molecules produced by immune cells, including T cells, whose plasma concentrations can reflect both the magnitude and nature of the immune response. Measuring plasma cytokine levels is thus a valuable approach for assessing immune system activity, especially in determining the functional state of T lymphocytes. A significant decrease in plasma concentrations of proinflammatory cytokines such as IL‐6, IFN γ, and TNFα, compared with the control group was observed following P. nigrum treatment (Figure 7A–C). Then, LY294002 treatment also reduced TNFα concentration (Figure 7C). In contrast Dox treatment decreased both pro‐inflammatory IL‐6 and TNFα, but also IL‐2 and IL‐4 cytokines (Figure 7A–D). No significant changes were observed for IL‐10 with any of the treatments (Figure 7F). These results suggest that P. nigrum extract can modulate immune response having a direct antitumor activity.

FIGURE 7.

Plasma cytokine levels in AML mice treated. (A) IL‐6, (B) IFN γ, (C) TNFα, (D) IL‐2, (E) IL‐4, and (F) IL‐10. In all cases, five samples per group were included, as this sample came from the plasma of mice via cardiac puncture at the time of euthanasia, and in several animals, it was not possible to obtain a good sample, or we obtained hemolyzed plasma that we could not analyze. Data are represented as the mean ± SEM. p values were calculated using the Mann–Whitney U‐test. **p < 0.01.

3.7. The Effector Profile of T Cells Is Modulated by the P. nigrum Extract

In the context of AML, as for other tumors, generating an effective and long‐lasting antitumor response is essential to control not only the primary tumor but also metastases. One key pathway involved in immune regulation is the PI3K/AKT/mTOR signaling cascade, which plays a pivotal role in T cell differentiation by favoring the generation of effector memory T cells (TEM), known for their effectiveness in long‐term immune surveillance, over terminally differentiated effector memory T cells (TEMRA), which exhibit reduced efficacy in the long term (Pompura and Dominguez‐Villar 2018). Since P. nigrum has activity on this pathway in tumor cells, we evaluated the role of this extract also on the T cells generated in the murine model of AML.

Our results revealed a significant increase in the percentage of splenic CD3⁺ T cells in the LY294002‐treated group compared with untreated animals (Figure 8A), while no statistically significant differences were observed in the total CD8⁺ T cell population among the experimental groups (Figure 8B).

FIGURE 8.

Immunomodulatory effects of Piper nigrum extract on splenic CD8⁺ and CD4⁺ T cell subsets and memory phenotypes (A) Percentage of total CD3⁺ T cells. (B) Frequency of CD8⁺ T cells within the CD3⁺ population. (C) Expression of exhaustion markers TIM‐3 and PD‐1 on CD8⁺ T cells. (D) Distribution of memory subsets within CD8⁺ T cells: Naïve T_N (CD62L+, CD44−), central memory T_CM (CD62L+, CD44+), effector memory T_EM (CD62L−, CD44+), and terminal effector memory T_EMRA (CD62L−, CD44‐). (E) Frequency of CD4⁺ T cells within the CD3⁺ population. (F) Percentage of CD4⁺ regulatory T cells (Tregs). (G) Expression of TIM‐3 and PD‐1 on CD4⁺ T cells. (H) Distribution of memory subsets within CD4⁺ T cells. The number of samples analyzed differs between experimental groups because the following data were generated from frozen splenic samples prepared at euthanasia and thawed for T cell isolation. Post‐thawing, the cell viability was lower than 30% in some cases, leading to elimination of such samples from further processing. Prevalence ratios were estimated with 95% CI, and p values were made by a Mann–Whitney U test. p < 0.05. p values were calculated using a Mann–Whitney U test. *p < 0.05.

Since P. nigrum treatment was shown to modulate the PI3K/AKT/mTOR pathway by decreasing Gal‐9 expression in leukemic tumor cells, we investigated whether this modulation could influence the expression of inhibitory immune checkpoint markers, such as Tim‐3 and PD‐1, on CD8 T cells. However, no significant differences in the co‐expression of these markers were observed across any of the treatments compared with the PBS control group (Figure 8C).

Upon further analysis of CD8 T cell subpopulations, a significant reduction in T_EMRA cells compared with the PBS control group, with a significant increase in T_EM cells was evidenced after P. nigrum , LY294002, and doxorubicin treatment. Then, doxorubicin also promotes a notable increase in central memory T cells (T_CM) (Figure 8D).

In contrast to the effects observed on CD3⁺ and CD8⁺ T cell compartments, treatment with P. nigrum did not significantly alter the overall frequency of CD4⁺ T cells compared with the PBS control (Figure 8E). Furthermore, while both doxorubicin and the PI3K inhibitor LY294002 significantly increased the proportion of regulatory CD4⁺ T cells (CD4⁺ Tregs), P. nigrum did not induce a comparable expansion of this immunosuppressive subset (Figure 8F), suggesting a differential immunomodulatory profile.

Importantly, P. nigrum treatment did not lead to upregulation of exhaustion markers TIM‐3 and PD‐1 on CD4⁺ T cells, in contrast to the marked increase induced by doxorubicin (Figure 8G). This indicates that P. nigrum , unlike conventional cytotoxic agents, may preserve T cell functionality by avoiding the induction of an exhausted phenotype.

At the level of CD4⁺ T cell memory subsets, a notable increase in the proportion of effector memory T cells (T_EM) was observed following treatment with P. nigrum , LY294002, and doxorubicin, compared with the PBS group (Figure 8H). This shift suggests that P. nigrum may enhance T cell differentiation toward an effector memory phenotype without compromising cell viability or promoting exhaustion, which could have implications for the design of combinatory immunotherapeutic strategies.

4. Discussion

AML is a diverse hematologic malignancy in which immature myeloid cells proliferate uncontrollably, transforming  the hematopoietic microenvironment to enhance disease progression. Despite the progress made in elucidating the genetic and epigenetic mechanisms underlying AML, treatment options are scarce and frequently not curative. The treatment that consists of aggressive  chemotherapy and hematopoietic stem cell transplantation fails to offer universal cure. Disease control is  not effective in most cases, particularly for the relapsed patients or those who have high‐risk features (da Rocha Silla et al. 2013; Wang and Baron 2020).

Due to these limitations, new therapeutic approaches are being developed where the objective is not only to inhibit  the growth of tumor cells as in classical cytotoxic agents, but also to reactivate or enhance response which is impaired by both disease and chemotherapy. A promising alternative in this setting is the polymolecular drugs derived from natural products, including plant extracts, have emerged as promising, since evidence indicates that these compounds could have an impact on antitumor response modulation; thus, they may have effects in AML treatment (Reilly et al. 2013; Moody et al. 2020).

In this study, P. nigrum extract was shown to have significant antitumor activity in an AML model. This effect appears to be associated with the inhibition of the PI3K/AKT/mTOR pathway, a key signaling cascade involved in the survival, proliferation, and glycolytic metabolism of tumor cells. By reducing PI3K and AKT phosphorylation, P. nigrum extract blocks mTOR signaling, resulting in a cytotoxic effect on leukemia cells.

Although the present results show that P. nigrum treatment reduces both PI3K/AKT/mTOR phosphorylation and the proportion of metabolically active tumor cells, these measurements were performed at a single time point (24 h). Therefore, the temporal relationship between pathway inhibition and metabolic reprogramming could not be determined. Future studies incorporating time‐course analyses will be necessary to establish the sequence of these molecular and metabolic events.

A particularly relevant finding of this study is the possible involvement of the PI3K/AKT/mTOR pathway not only in tumor metabolism but also in the regulation of Gal‐9 expression. This relationship has been documented in the literature through the activation of NF‐kB, contributing to the establishment of an immunosuppressive tumor microenvironment (Le Chen et al. 2022; Kandel et al. 2021). The Overexpression of Gal‐9 in tumor cells contributes to immune evasion by promoting the survival of malignant cells and inactivating the function of NK cells and CD8 T lymphocyte (Mafi et al. 2022). In this study, we observed that in vitro treatment with P. nigrum achieved a decrease in glycolytic metabolism and Gal‐9 expression in DA‐3/ER‐GM tumor cells, and interestingly, in vivo, mice with AML treated with this extract showed lower tumor growth, longer survival, a significant increase in memory effector T cells (T_EM), and a reduction in terminally differentiated memory effector T cells (T_EMRA). These findings suggest that the extract possibly achieves a reduction in Gal‐9 in vivo, which improves a more effective immune and memory response, while preventing an exhausted effector response in the in vivo model.

From an immunological perspective, P. nigrum also influences the tumor microenvironment by promoting the infiltration and activation of immune cells, such as NK cells, cytotoxic T lymphocytes (particularly CD8 TL), and dendritic cells, while reducing myeloid suppressor cells and regulatory T cells (T_regs), as demonstrated in other tumor model (Lasso et al. 2023). This immunomodulatory effect is particularly relevant in combination therapies, where enhanced immune surveillance may improve overall antitumor response. Furthermore, P. nigrum 's ability to mitigate chronic inflammation by regulating proinflammatory cytokines, such as IL‐6 and TNF‐α, further underscores its potential to counteract tumor‐associated immunosuppression (Récher 2021; Ahmed et al. 2023).

An emerging aspect that has not been explored in this study is the ability of P. nigrum to induce immunogenic cell death (ICD). Piperine has been reported to induce ER stress and have a clear pro‐oxidative effect, both of which are key factors in the induction of ICD (Guo et al. 2021). This form of cell death is characterized by the exposure or release of danger‐associated molecular patterns (DAMPs), such as calreticulin, ATP, and HMGB1, which are essential for dendritic cell maturation and antigen presentation to T cells. ER stress‐induced ICD transforms dying tumor cells into a source of tumor antigens capable of enhancing adaptive immune responses, a highly desirable property in leukemia models, where immune evasion predominates.

In this context, doxorubicin was included in our study as a clinically relevant therapeutic comparator, not only because of its well‐established cytotoxic activity but also because anthracyclines are recognized as potent immunomodulatory agents capable of inducing ICD. Doxorubicin promotes the exposure or release of DAMPs, including calreticulin, ATP, HMGB1, annexin A1, and type I interferon‐related signals, thereby enhancing dendritic cell maturation, antigen presentation, and adaptive antitumor immunity. Furthermore, previous studies have demonstrated that the therapeutic efficacy of pegylated liposomal doxorubicin (Doxil) depends, at least in part, on host immunity. In immunocompetent mice, Doxil increased tumor immunogenicity through enhanced MHC‐I expression while reducing regulatory T cells, tumor‐associated macrophages, and myeloid‐derived suppressor cells, thereby promoting T‐cell‐mediated antitumor responses (Takayama et al. 2020). In addition, doxorubicin exerts direct cytotoxic effects through ROS generation, inducing oxidative damage, apoptosis, and necrosis in leukemia cells, effects that are markedly attenuated by antioxidant treatment (Szwed et al. 2015). These reports support our observations, in which doxorubicin‐treated animals exhibited reduced leukemic burden, partial preservation of hematological parameters, and modulation of T‐cell memory subsets. Nevertheless, the antileukemic effects observed with P. nigrum were generally comparable and, for some parameters, more pronounced than those obtained with doxorubicin or the PI3K inhibitor LY294002.

Together, these findings suggest that P. nigrum may exert antileukemic activity through complementary mechanisms involving inhibition of the PI3K/AKT/mTOR pathway, metabolic reprogramming, oxidative stress induction, and immune modulation. However, since biomarkers of ICD, oxidative damage, and functional changes in immunosuppressive myeloid populations were not evaluated in the present study, future investigations should determine the relative contribution of these mechanisms to the overall therapeutic activity of P. nigrum in AML.

Moreover, we observed a decrease in TEMRA and an increase of TEM upon P. nigrum treatment. This may relate to regulation of the  PI3K/AKT/mTOR pathway that is critical for control of T cell differentiation, metabolism, and fate determination. In particular, high PI3K activity is linked with T effector cell differentiation and exhaustion whereas its suppression promotes memory cell formation, metabolic plasticity as well as long‐term persistence, which are key components of successful antitumor immunity (Nepstad et al. 2020). Mechanistically, this shift could be attributed to the compromised mTORC1 signaling, Blimp‐1  downregulation and TCF‐1 preservation in multipotent/stem cell‐like states of T cells (Mafi et al. 2022). Thus, PI3K suppression in T cells is an intrinsic metabolic checkpoint to provide them with superior functional fitness and robustness for persistent immune surveillance against leukemic cells and thus optimal immunotherapeutic benefits (Feng et al. 2023).

Beyond AML, the bioactive compounds of P. nigrum , particularly piperine, have demonstrated antitumor activity in various solid and hematological cancers (Wu et al. 2023; Turrini et al. 2020). This activity is achieved by modulating key signaling pathways, such as NF‐kB, JAK/STAT, and MAPK, which are critically involved in tumor progression, immune evasion, and treatment resistance (Ginzburg et al. 2014). Furthermore, P. nigrum extract has been shown to reduce glucose uptake in tumor cell lines such as 4T1, leading to decreased tumor growth in vivo, findings consistent with observations made in this AML model (Lasso et al. 2023). Interestingly, other studies have reported that P. nigrum may enhance the efficacy of chemotherapy by increasing drug bioavailability and reducing multiple drug resistance mechanisms in tumor cells (Wiraswati et al. 2025).

Importantly, the concentrations used in vitro exhibited selective cytotoxicity against leukemic cells, with no vehicle‐associated toxicity observed, as ethanol had no effect on cell viability at the tested concentrations. In vivo, the selected dose was based on tolerability and previously reported bioactive ranges. Animals treated with the extract did not exhibit significant weight loss, behavioral alterations, or clinical signs suggestive of systemic toxicity, which supports a favorable therapeutic window under the experimental conditions. Nevertheless, these findings are limited to acute toxicity, and comprehensive long‐term toxicological studies, including repeated‐dose toxicity, hematological, biochemical, and histopathological analyses, are necessary to fully establish the safety profile of the extract prior to clinical translation.

Regarding translational considerations, although direct extrapolation from murine models to humans requires caution, dose conversion based on body surface area normalization suggests that the effective dose observed in this study may fall within a range potentially achievable in humans. However, pharmacokinetic characterization, including absorption, distribution, metabolism, and excretion (ADME), together with oral bioavailability studies and formal dose‐escalation evaluations are required before clinical translation can be considered.

The magnitude of the in vivo effect, including reduced tumor progression and improved survival in an aggressive AML model, represents a biologically meaningful outcome. While the extract did not replace the efficacy of doxorubicin, its immunomodulatory properties and metabolic effects suggest potential value as an adjunct therapy. The ability to enhance effector memory T cell responses while limiting terminal differentiation may be particularly relevant in the context of relapse prevention and long‐term disease control.

Although plant‐derived compounds are often administered orally, the intraperitoneal route was selected in this proof‐of‐concept study to ensure controlled systemic exposure, reproducible dosing, and to avoid variability associated with gastrointestinal absorption and first‐pass metabolism. Future studies should evaluate oral formulations to better simulate potential clinical use and assess bioavailability constraints.

Several limitations of the present study should be acknowledged. First, although inhibition of the PI3K/AKT/mTOR pathway and reduced Gal‐9 expression were demonstrated in vitro, these molecular targets were not directly evaluated in leukemic tissues from treated animals. Therefore, the proposed mechanistic link between pathway inhibition and the observed in vivo antileukemic effects remains indirect and should be confirmed in future studies. Second, phosphorylation of the PI3K/AKT/mTOR pathway and metabolic parameters were evaluated at a single time point, preventing determination of the temporal sequence between pathway inhibition and metabolic reprogramming. Third, although cytotoxicity and increased intracellular ROS production were observed, direct evaluation of apoptosis was not performed, and therefore the specific mode of tumor cell death remains to be characterized. Finally, pharmacokinetic characterization and comprehensive toxicological analyses, including histopathological evaluation and long‐term safety assessment, were beyond the scope of this study. Future investigations addressing these aspects will be essential to further elucidate the mechanisms of action and define the translational potential of P. nigrum extract in AML.

5. Conclusion

In summary, P. nigrum extract demonstrated significant antileukemic activity both in vitro and in the DA3ER murine AML model, associated with inhibition of the PI3K/AKT/mTOR pathway, reduced glycolytic metabolism, and modulation of immunoregulatory pathways. These effects included a decrease in Gal‐9 expression and a favorable shift in T‐cell memory subtypes, along with a reduction in circulating blast burden, decreased infiltration into hematopoietic organs, and partial preservation of hematopoiesis in vivo.

It is important to note that the administered dose (15.67 mg/kg) was approximately four times lower than the LD₅₀ calculated in BALB/c mice and did not induce observable signs of acute toxicity, supporting a favorable therapeutic window. The reduction in tumor burden and prolonged survival observed in treated animals further reinforces the potential of P. nigrum as a complementary therapeutic strategy in AML.

However, some limitations of this study should be acknowledged. Although the extract modulated PI3K/AKT/mTOR signaling and Gal‐9 expression under controlled in vitro conditions, no direct molecular validation of these mechanisms was performed in leukemic tissues in vivo. Therefore, although the convergence between in vitro mechanistic findings and in vivo phenotypic results strongly supports the biological relevance of this signaling pathway, additional studies are required to establish a direct causal relationship under in vivo conditions.

Future research should include the evaluation of PI3K/AKT/mTOR pathway activation and Gal‐9 expression in tumor and hematopoietic tissues, as well as the implementation of more advanced mechanistic approaches, including models of genetic or pharmacological inhibition of the pathway, in order to further validate the molecular targets involved in the antileukemic activity of P. nigrum . Additionally, pharmacokinetic and long‐term toxicity studies will be necessary to support its future translational development.

Author Contributions

Claudia Urueña: conceptualization, investigation, writing – original draft, methodology, validation, formal analysis, supervision, writing – review and editing. Paola Lasso: conceptualization, investigation, methodology, validation, writing – original draft, formal analysis, data curation, writing – review and editing. Susana Fiorentino: conceptualization, funding acquisition, investigation, writing – original draft, writing – review and editing, visualization, supervision. Natalia Murillo: methodology, writing – original draft, formal analysis, data curation.

Funding

This work was supported by the Pontificia Universidad Javeriana (FP44842‐221‐2018), the Ministerio de Ciencia, Tecnología e Innovación (FP44842‐221‐2018), the Ministerio de Educación de la Nación (FP44842‐221‐2018), the Ministerio de Comercio, Industria y Turismo (FP44842‐221‐2018), the Instituto Colombiano de Crédito Educativo y Estudios Técnicos en el Exterior (FP44842‐221‐2018), and the World Bank Group (FP44842‐221‐2018).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Representative culture images of ethanol‐treated control cells (upper) compared to Piper nigrum extract treated cells (lower).

Figure S2: Representative flow cytometry analysis of phosphorylated proteins in tumor cells under different treatments at 24 h. Representative flow cytometry plots are shown for the detection of phosphorylated proteins of the PI3K/AKT/mTOR pathway under three experimental conditions: basal (black), P. nigrum extract treatment (aquamarine), and LY294002 treatment (dark blue). The analysis was performed using a sequential gating strategy. First, cells were selected based on size and granularity using FSC‐A versus SSC‐A. Subsequently, viable cells were identified using a viability dye (AQUA). From this population, metabolic subpopulations were defined according to signal intensity (Hi, Int, and Low). Finally, within the Hi subpopulation characterized by higher metabolic activity, proliferative capacity, and cellular complexity, the frequency of cells positive for phosphorylated PI3K (PE), phosphorylated AKT (APC), and phosphorylated mTOR (PE‐Cy7) was evaluated for each experimental condition.

Figure S3: Flow cytometry analysis of phosphorylated proteins in tumor cells under different treatments at 6 h. Representative flow cytometry plots are shown for the detection of phosphorylated proteins of the PI3K/AKT/mTOR pathway under three experimental conditions: basal (black), P. nigrum extract treatment (aquamarine), and LY294002 treatment (dark blue). The analysis was performed using a sequential gating strategy. First, cells were selected based on size and granularity using FSC‐A versus SSC‐A. Subsequently, viable cells were identified using a viability dye (AQUA). From this population, metabolic subpopulations were defined according to signal intensity (Hi, Int, and Low). Finally, within the Hi subpopulation characterized by higher metabolic activity, proliferative capacity, and cellular complexity, the frequency of cells positive for phosphorylated PI3K (PE), phosphorylated AKT (APC), and phosphorylated mTOR (PE‐Cy7) was evaluated for each experimental condition.

Table S1: Information on all antibodies used.

Table S2: Quantitative analysis of metabolic subpopulations and phosphorylated proteins and fold change calculation at 24 h. This table summarizes the frequency values of the previously described metabolic subpopulations (Hi, Int, and Low) and phosphorylated proteins (pPI3K, pAKT, and pmTOR) obtained by flow cytometry at 24 h under basal conditions, P. nigrum treatment, and LY294002 treatment. Basal conditions were used as the reference (set to 1) to calculate fold change values, allowing comparison of treatment‐induced effects relative to baseline levels. Fold change was calculated by normalizing each experimental condition to the average basal value for each parameter. The table shows both the raw frequency data and the corresponding fold change values for each treatment condition. Data presented correspond to one representative experimental replicate; however, the same trend was consistently observed across all independent experiments performed.

Table S3: Quantitative analysis of metabolic subpopulations and phosphorylated proteins and fold change calculation at 6 h. This table summarizes the frequency values of the previously described metabolic subpopulations (Hi, Int, and Low) and phosphorylated proteins (pPI3K, pAKT, and pmTOR) obtained by flow cytometry at 6 h under basal conditions, P. nigrum treatment, and LY294002 treatment. Basal conditions were used as the reference (set to 1) to calculate fold change values, allowing comparison of treatment‐induced effects relative to baseline levels. Fold change was calculated by normalizing each experimental condition to the average basal value for each parameter. The table shows both the raw frequency data and the corresponding fold change values for each treatment condition. Data presented correspond to one representative experimental replicate; however, the same trend was consistently observed across all independent experiments performed.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.