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. 2025 Dec 22;15:45058. doi: 10.1038/s41598-025-32898-8

Anticancer effect of piperine, a black pepper compound, regulating apoptosis mediated through extracellular vesicles and cathepsin D in acute leukemia

Kantorn Charoensedtasin 1, Wasinee Kheansaard 1, Sittiruk Roytrakul 2, Dalina Tanyong 1,✉
PMCID: PMC12749100  PMID: 41423483

Abstract

Extracellular vesicles (EVs) contribute to tumorigenesis in acute leukemia, while natural compounds can counteract these EVs-mediated mechanisms. Piperine, a natural black pepper compound, exerts anticancer activity toward many cancer cells with minimal effect in normal PBMC cells via the apoptosis signaling pathway, a key programmed cell death pathway. The aim of this study was to investigate the involvement of EVs in piperine-induced apoptosis in acute leukemia. NB4 and MOLT-4 leukemic cell lines were treated with various concentrations of piperine. Cell viability, apoptosis, and cell cycle distribution were assessed using MTT assays and flow cytometry, respectively. Bioinformatic analysis was utilized to identify EV-related target proteins of piperine. RT–qPCR and Western blotting were performed to evaluate the expression of proapoptotic genes and proteins, respectively. Our findings demonstrated that piperine inhibited cell proliferation and induced apoptosis in both leukemic models. Cathepsin D (CTSD) has emerged as a piperine-responsive protein which was regulated apoptosis. Notably, EVs from piperine-treated leukemic cells upregulated CTSD and proapoptotic genes and proteins, Bax and caspase-3. These findings suggest that piperine modulates EV-mediated communication to promote apoptosis in acute leukemia. Interestingly, this work highlights the potential of piperine as a natural therapeutic agent targeting EV-mediated leukemic apoptosis.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-32898-8.

Keywords: Piperine, Apoptosis, Extracellular vesicles, Cathepsin d, Acute leukemia

Subject terms: Cancer, Cell biology, Molecular biology

Introduction

Acute leukemia is a hematological malignancy characterized by rapid, unusual excess leukocyte proliferation, consequently interfering with the production of normal hematopoietic cells; this causes some complications in leukemic patients, such as anemia or thrombocytopenia1. The trends of acute leukemia incidence and death have slightly increased every year over the last ten years. Acute leukemia is considered the most difficult form of treatment among hematological malignancies2. There are several treatment approaches for leukemia, including chemotherapy; immunotherapy, such as the use of cytokine or CAR–T cells; and allogenic transplantation. However, these approaches have many side effects and high costs3,4. Complementary and alternative medicines, especially natural compounds, could be utilized for cancer prevention and treatment because they are safe, have fewer side effects, and are cost effective. Moreover, natural compounds can be used with chemotherapy to increase the likelihood of recovery in patients and decrease chemotherapy doses5. Therefore, herbal medicines could be used as alternative medicines or combined with other chemical drugs.

Piperine is a major compound extracted from black pepper (Piper nigrum), the fruit of which is spicy, and it has many useful pharmacological effects, including antioxidant, antimicrobial, and anticancer effects6,7. Moreover, many studies have revealed the anticancer effects of piperine mediated via various signaling pathways, such as the PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways8–10. Inducing apoptosis-mediated cell death is a well-known strategy for eliminating cancer cells by targeting molecules in signaling pathways to induce the caspase cascade11. Most natural compounds that induce cancer cell apoptosis interact with apoptotic proteins in signaling pathways12.

Extracellular vesicles (EVs), small molecular carriers, play a vital role in promoting cancer cell growth and supporting the survival of cancer cells. In normal situations, cancer cells release EVs to transfer biological molecules, including mRNAs, miRNAs, and proteins, via endocytosis to terminal cells, and these biological molecules can enhance or suppress signaling pathways. EVs released from leukemia have essential roles to promote leukemia progression by transferring biological molecules to leukemic cells or supporting in microenvironment13. This phenomenon can possibly inhibit programmed cell death and promote multidrug resistance in acute leukemia14. Importantly, unique external stimuli can stimulate individual biological molecules in EVs. For example, hypoxia increases the secretion of EVs carrying proteins and miRNAs such as miR-210 and miR-135b, which are involved in angiogenesis and metastasis15,16. Additionally, some natural compounds affect biological molecules in EVs, and natural compound-treated EVs can inhibit cancer cell growth17.

From a previous study, cathepsin D (CTSD) level was increased in EV of leukemic cell lines18. Cathepsin D (CTSD) has been reported to be involved in several cancer proliferations and apoptosis19. Although several studies have investigated the anticancer effect of piperine in many types of cancer cells20,21, the effect of piperine on EV-mediated apoptosis via cathepsin D (CTSD) in acute leukemia has less evidence. Therefore, the aim of this study was to investigate the anticancer effects of piperine on the apoptosis signaling pathway mediated by EVs in acute leukemia.

Results

Inhibition of NB4 and MOLT-4 cell proliferation by piperine

The structure of piperine is shown in Fig. 1A. The effect of piperine on NB4 and MOLT-4 cell proliferation was investigated using the MTT assay. NB4 and MOLT-4 cell proliferation was significantly inhibited by piperine at 24 h and 48 h. However, it exhibited less effect on PBMCs (Fig. 1B). After 24 h, the IC50 values of piperine were 224 µM in NB4 cells and 384 µM in MOLT-4 cells; at 48 h, the IC50 values were 145 µM and 156 µM, respectively. Therefore, piperine inhibits NB4 and MOLT-4 cell proliferation in a dose- and time-dependent manner, with minimal effects on PBMCs. Since IC50 at 48 h value was less than IC50 at 24 h value in NB4 and MOLT-4 cells and the concentration of piperine from previous study22, IC50 was calculated and the lowest IC50 at 48 h was chosen for use in further study in both NB4 and MOLT-4 cells.

Fig. 1.

Fig. 1

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Inhibitory effect of piperine in NB4 and MOLT-4 cells in dose-dependent manners for 24 h and 48 h. (A) Chemical structure of piperine compound. (B) NB4, MOLT-4 and normal PBMC were treated with 0, 50, 100, and 200 µM for 24 h and 48 h, and then cell viability of NB4, MOLT-4 and normal PBMC was analyzed using MTT assay. The three independent data were represented as mean ± standard error of mean (S.E.M) (n = 3). The statistical comparison was performed using one-way ANOVA. * p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 were considered as significant difference.

Synergistic effects of piperine and daunorubicin on NB4 and MOLT-4 cells

The viability of NB4 and MOLT-4 cells treated with piperine and 0.2 µM daunorubicin, a chemotherapeutic drug for leukemia treatment, was investigated via the MTT assay. CompuSyn software was used to measure the combination index. The viability of NB4 and MOLT-4 cells treated with combination piperine and daunorubicin was significantly decreased compared to control or piperine or daunorubicin alone (Fig. 2A). Moreover, the combination index (CI) of piperine and daunorubicin was analyzed by CompuSyn (Supplementary Figure S1). The average CI values of combination piperine and daunorubicin were 0.59 and 0.81 in NB4 and MOLT-4 cells, respectively (Fig. 2B). These findings indicate a synergistic effect from the combination of piperine and daunorubicin.

Fig. 2.

Fig. 2

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Combination of piperine and daunorubicin is exhibited as synergistic effect in NB4 and MOLT-4 cells. (A) NB4 and MOLT-4 cells were treated with IC50 of piperine, 0.2 µM of daunorubicin and combination of piperine and daunorubcin for 48 h and then cell viability of NB4 and MOLT-4 cells was analyzed using MTT assay. (B) A combination index plot of combination of piperine and daunorubicin was constructed using CompuSyn. Fa (fraction affected) indicated the effect of combination of piperine and daunorubicin of three independent experiments. CI indicated combination index of each independent experiment. “P” was a piperine compound and “D” was a daunorubicin compound. The data were represented as mean ± standard error of mean (S.E.M) (n = 3). The statistical comparison was performed using one-way ANOVA. *** p < 0.001 and **** p < 0.0001 were considered as significant difference.

Apoptotic induction by piperine

NB4 and MOLT-4 cells were treated with the IC50 of piperine for 48 h, and apoptosis was assessed by Annexin V-conjugated FITC and propidium iodide (PI) staining and flow cytometry. Compared with the control, piperine significantly increased the percentage of apoptotic NB4 and MOLT-4 cells (Fig. 3A) (Supplementary Figure S5). To confirm the induction of apoptosis by piperine, the cell cycle distribution of piperine-treated NB4 and MOLT-4 cells was investigated via PI staining and flow cytometry. Piperine significantly increased the percentages of both NB4 and MOLT-4 cells in the sub-G1 stage, which indicated the DNA fragmentation of cells from piperine treatment (Fig. 3B) (Supplementary Figure S6).

Fig. 3.

Fig. 3

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Piperine induces apoptosis in NB4 and MOLT-4 cells. NB4 and MOLT-4 cells were treated with IC50 piperine for 48 h and then apoptosis and cell cycle of NB4 and MOLT-4 cells were analyzed using flow cytometry. (A) Scatter plot of apoptosis of NB4 and MOLT-4 after treating piperine. Q1: Annexin V +/PI -; Q2: Annexin V +/PI +; Q3: Annexin V -/PI -; Q4: Annexin V +/PI -. Percentage of apoptosis of NB4 and MOLT-4 cells after treating piperine. (B) Histogram of cell cycle of NB4 and MOLT-4 cells after treating piperine. Percentage of sub G1 of total NB4 and MOLT-4 cells after treating piperine. The data were represented as mean ± standard error of mean (S.E.M) (n = 3). The statistical comparison was performed using an unpaired t test. ** p < 0.01 were considered as significant difference.

Cathepsin D is a piperine–responsive protein involved in EVs

The STITCH and ChEMBL databases were utilized to predict possible piperine-responsive proteins. As depicted in Supplementary Table S1, eighteen and twenty-one proteins were predicted from STITCH and ChEMBL, respectively. Thus, thirty-nine total piperine-responsive proteins were predicted from the 2 databases. The proteomic profile of EVs from a previous study23 was filtered to include only leukemia samples, revealing 1296 leukemic EV proteins (Supplementary Table S2). Two of these proteins, cathepsin D (CTSD) and Niemann‒Pick C1 (NPC1), were also predicted as piperine-responsive proteins (Fig. 4A). Additionally, a protein–protein interaction (PPI) network was constructed via STRING. The results revealed that only CTSD interacted with CASP3 and BID, whereas NPC1 interacted with only CTSD (Fig. 4B). The combined interaction score between CTSD and CASP3 was 0.568, whereas that between CTSD and BID was 0.917. These findings suggest that a possible target protein of piperine involved in EVs and the apoptosis is CTSD.

Fig. 4.

Fig. 4

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Bioinformatic tools predict cathepsin D (CTSD) as piperine-responsive target proteins involved in extracellular vesicles (EVs). (A) A draw Venn-diagram predicted CTSD and NPC1 as piperine-responsive proteins in EVs using STITCH/ChEMBL and a proteomic profile of leukemic EV. (B) Protein-protein interaction constructed by STRING demonstrated piperine-responsive proteins involved in EVs and apoptotic proteins including CASP3, BID and BAX.

Confirm cathepsin D, a target protein of piperine, using pepstatin A

In this study, pepstatin A, a CTSD inhibitor, was used to confirm the role of CTSD involved in apoptosis24. NB4 and MOLT-4 cells were treated with 10 µM of pepstatin A for 24 h. After that, NB4 and MOLT-4 cells were treated with IC50 of piperine for 48 h and then detected using flow cytometry. The results showed that pepstatin A significantly reduced percentage of piperine-induced apoptosis in NB4 and MOLT-4 cells compared to treatment with piperine alone (Fig. 5) (Supplementary Figure S7). This result indicates that CTSD might be involved in piperine-induced apoptosis in NB4 and MOLT-4 cells.

Fig. 5.

Fig. 5

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Pepstatin A, a CTSD inhibitor, reduces percentage of piperine-induced apoptosis in NB4 and MOLT-4 cells. NB4 and MOLT-4 cells were pre-treated with pepstatin A for 24 h. After that, NB4 and MOLT-4 cells were treated with IC50 piperine for 48 h and then NB4 and MOLT-4 cells were detected using flow cytometry. Scatter plot of apoptosis of NB4 and MOLT-4 after treating piperine and pepstatin A. Q1: Annexin V +/PI -; Q2: Annexin V +/PI +; Q3: Annexin V -/PI -; Q4: Annexin V +/PI -. Percentage of apoptosis of NB4 and MOLT-4 cells after treating piperine and pepstatin A. The data were represented as mean ± standard error of mean (S.E.M) (n = 3). The statistical comparison was performed using one-way ANOVA. ** p < 0.01 and **** p < 0.0001 were considered as significant difference.

Apoptotic gene and protein expression is regulated by piperine in NB4 and MOLT-4 cells

NB4 and MOLT-4 cells were treated with the IC50 of piperine, and the levels of CTSD, apoptotic genes and proteins, including Bax and caspase–3, were assessed using RT–qPCR and Western blotting. The gene expression results revealed that the relative mRNA expression levels of CTSD, Bax, and Casp3 in NB4 and MOLT-4 cells were significantly greater in the piperine treatment group than in the control group (Fig. 6A). The protein expression results revealed that CTSD, Bax, and caspase-3 were significantly increased after piperine treatment in NB4 and MOLT-4 cells (Fig. 6B) (Supplementary Figure S2). These findings indicate that piperine can induce the apoptosis by upregulating CTSD in NB4 and MOLT-4 cells.

Fig. 6.

Fig. 6

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Piperine increases CTSD and pro-apoptotic genes and proteins expression including Bax and caspase-3 in NB4 and MOLT-4 cells. NB4 and MOLT-4 cells were treated with IC50 piperine for 48 h and mRNA and proteins expression were determined using RT–qPCR and Western blotting, respectively. (A) Relative mRNA expressions include CTSD, Bax and casp3 genes in NB4 and MOLT-4 cells after treating piperine. (B) Band intensity and relative protein expressions include CTSD, Bax and caspase-3 proteins in NB4 and MOLT-4 cells after treating piperine. The data were represented as mean ± standard error of mean (S.E.M) (n = 3). The statistical comparison was performed using an unpaired t test. * p < 0.05, ** p < 0.01 and *** p < 0.001 were considered as significant difference.

Characterization of Con-EVs and Pip-EVs

NB4 and MOLT-4 cells were treated with the IC50 of piperine, and then supernatants were collected, filtered, and centrifuged at high speed to obtain piperidine-treated (Pip-EVs) and control (Con-EVs) EVs. Con-EV was defined as EVs release from untreated NB4 and MOLT-4 cells while Pip-EV was defined as EVs release from piperine-treated NB4 and MOLT-4 cells. After EVs were resuspended in PBS, Con-EVs and Pip-EVs were characterized via transmission electron microscopy (TEM), tunable resistive pulse sensing (TRPS), and Western blotting. The morphologies of Con-EVs and Pip-EVs are shown in Fig. 7A. The size and number of Con-EV and Pip-EV particles were determined via TRPS. The mean sizes of Con-EVs and Pip-EVs from NB4 cells were 301 ± 19 nm and 298 ± 8 nm, respectively, whereas the sizes of Con-EVs and Pip-EVs from MOLT-4 cells were 286 ± 2 nm and 302 ± 8 nm, respectively (Fig. 7B). Compared with the control, piperine significantly increased EV release from both NB4 and MOLT-4 cells (Fig. 7C). The International Society for Extracellular Vesicles (ISEV) suggested utilizing biomarkers for identifying EVs including tetraspanin (CD9 and CD63) and cytosolic (flotillin-1) proteins25. The membrane proteins and cytosolic proteins of Con-EVs and Pip-EVs were characterized via Western blotting, revealing the presence of CD9, CD63, and flotillin-1 proteins (Fig. 7D) (Supplementary Figure S3). Therefore, Con-EVs and Pip-EVs derived from NB4 and MOLT-4 cells were utilized in further experiments.

Fig. 7.

Fig. 7

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Characterization of Con-EV and Pip-EV from NB4 and MOLT-4 cells. NB4 and MOLT-4 cells were treated with IC50 piperine and then supernatant was collected to isolate EVs using 0.45 µM filter and high-speed centrifugation. After that, Con-EV and Pip-EVs were characterized using transmission electron microscopy, tunable pulse resist sensing (TRPS) and Western blotting. (A) Morphologies of EVs from control and piperine-treated NB4 and MOLT-4 cells. (B) Size distribution of EVs from control and piperine-treated NB4 and MOLT-4 cells. (C) The EVs particle number of control and piperine-treated NB4 and MOLT-4 cells. (D) CD9, CD63 and flotillin-1 markers of control and piperine-treated NB4 and MOLT-4 cells. The data were represented as mean ± standard error of mean (S.E.M) (n = 3). The statistical comparison was performed using an unpaired t test. * p < 0.05 and ** p < 0.01 were considered as significant difference.

Effects of Pip-EVs on the expression of apoptotic genes and proteins in NB4 and MOLT-4 cells

To understand how piperine affects NB4 and MOLT-4 cells through EVs, NB4 and MOLT-4 cells were treated with 10 µg/ml Con-EVs or Pip-EVs for 24 h, and then apoptotic genes and proteins expression were analyzed via RT‒qPCR and Western blotting, respectively. The relative mRNA expression levels of the CTSD, Bax, and Casp3 genes in NB4 and MOLT-4 cells were significantly greater in response to Pip-EVs than in response to Con-EVs (Fig. 8A). The relative protein expression levels of the CTSD, Bax, and caspase-3 proteins were also significantly greater in NB4 and MOLT-4 cells treated with Pip-EVs than in those treated with Con-EVs (Fig. 8B) (Supplementary Figure S4). These findings indicate that piperine can regulate the expression of apoptotic genes and proteins, including CTSD, Bax, and caspase-3, by interacting with EVs in NB4 and MOLT-4 cells, resulting in increased induction of the apoptosis signaling pathway.

Fig. 8.

Fig. 8

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Pip-EV enhances CTSD and pro-apoptotic genes and proteins expression including CTSD, Bax and caspase-3 in NB4 and MOLT-4 cells. NB4 and MOLT-4 cells were treated with 10 µg/ml Con-EV and Pip-EV for 24 h and then mRNA and protein expressions of NB4 and MOLT-4 cells were determined using RT–qPCR and Western blotting, respectively. (A) Relative mRNA expressions include CTSD, Bax and casp3 genes in NB4 and MOLT-4 cells after treating Con-EV and Pip-EV. (B) Relative protein expressions include CTSD, Bax and caspase-3 proteins in NB4 and MOLT-4 cells after treating Con-EV and Pip-EV. The three independent data were represented as mean ± standard error of mean (S.E.M) (n = 3). The statistical comparison was performed using an unpaired t test. * p < 0.05, ** p < 0.01 and **** p < 0.0001 were considered as significant difference.

Discussion

Acute leukemia is considered severe hematological malignancy due to plenty of complication and sophisticated treatment approaches26. Obviously, natural compounds could serve as an alternative treatment to cure leukemia and reduce the toxicity from chemotherapy27,28. For instance, gambogic acid, major compound in Garcinia hanburyi (gamboge) inhibited different leukemia such as T-ALL, AML or CML via apoptosis and PI3K/Akt/NF-ĸB signaling pathway29,30. Moreover, 6-methoxydihydroavicine, benzophenanthridine alkaloid induced caspase-mediated cell death in acute leukemia31. Our study emphasized piperine, a major black pepper compound, due to exhibiting various pharmacological effects including anti-cancer, anti-inflammatory and anti-microbial7,32 while exhibiting less effect in normal cells33. Additionally, a previous study exhibited a protective role of piperine by inhibiting oxidative stress in normal cells34. Although the anti-cancer effect of piperine in leukemic cells such as HL-60 and K562 have already investigated20–22, our study investigated the anti-leukemic effect of piperine in NB4 cells, acute promyelocytic leukemia, and MOLT-4 cells, T-acute lymphocytic leukemia. According to the results, piperine inhibited leukemic cell proliferation while exhibiting minimal cytotoxicity in normal peripheral blood mononuclear cells (PBMCs). Moreover, our study demonstrated the synergistic effects of combination of piperine and daunorubicin. Daunorubicin, an anthracycline antibiotic, is a chemotherapy against acute leukemia and chronic leukemia widely used in clinical treatment. Daunorubicin and other anthracycline antibiotics interacts with DNA between CG–GC base pairs causing DNA damage, leading to cell cycle arrest35. Piperine is a well-known compound for combining with other chemotherapies or other natural compounds. For example, piperine with gambolic acid synergistically induced apoptosis cell death in human cholangiocarcinoma cell36. In case of combination between piperine with chemotherapy, the use of piperine with doxorubicin synergistically induced triple-negative breast cancer MDA-MB-231 cells sensitivity via PI3K/Akt/mTOR pathway37.

In recent years, many natural compounds have been shown to target key proteins involved in apoptotic signaling. Piperine has also been reported as an apoptotic induction compound in many cancer cells38. Our findings demonstrated that piperine induced apoptosis in NB4 and MOLT-4 cells. Moreover, our bioinformatic tools identified a piperine-responsive target protein, cathepsin D (CTSD) in this study. To confirm CTSD as a piperine-responsive protein, pepstatin A, a CTSD inhibitor, was used to investigate the role of CTSD in apoptosis24,39. According to the result, pepstatin A reduced piperine-induced apoptosis in NB4 and MOLT-4 cells. Piperine induced CTSD and proapoptotic genes and proteins, including Bax, and caspase-3 in acute leukemia. CTSD is an aspartyl protease that mainly degrades proteins within lysosomes and requires an acidic pH for proper function19. Decreased CTSD is associated with many diseases, such as neurodegenerative disease. Breast cancer cells have high levels of extracellular CTSD, which induces invasion and metastasis by enhancing intercellular cell adhesion molecule-1 (ICAM-1)40,41. The protein‒protein interactions identified in this study revealed that CTSD can interact with BID and caspase-3 in the apoptosis signaling pathway. However, CTSD interacted with Bax indirectly. Recent reports showed that the release of CTSD from lysosomes induces both BID and Bax19,42,43. This study emphasized the apoptosis signaling pathway, specifically intrinsic pathway in NB4 and MOLT-4 cells induced by piperine. From protein‒protein interaction result, CTSD are associated with intrinsic pathway of apoptosis by inducing Bax. From Guo L et al., activation of apoptosis by piperine was associated with ROS related to mitochondria signaling pathway, consequently activating caspase-9 and caspase-3 in gastric cancer cells44. The overexpression of CTSD can increase apoptosis via the release of cytochrome c and the activation of the caspase cascade45. Interestingly, some natural compounds have demonstrated the role of CTSD in eliminating cancer cells. For example, Agrocybe cylindracea fucoglucogalactan, a polysaccharide compound, can induce colorectal cancer apoptosis via the CTSD lysosome‒mitochondria axis46. In another study, a natural phenolic resveratrol induced apoptosis and autophagy via release of CTSD from lysosome in drug-resistant leukemia47.

Extracellular vesicles (EVs) are important for their role in cell‒cell communication and ability to reprogram surrounding cells. Importantly, leukemic EVs cause dysfunction in normal cells and enhance the supporting function of the microenvironment48. Moreover, leukemic EVs which are treated with chemotherapy are transferred to reduce apoptosis in acute leukemic cells49. The use of natural compounds could be used to reduce this phenomenon. For a recent study, we firstly isolate EVs from piperine-treated leukemic cells (Pip-EV) using high-speed centrifugation and filtration to investigate their apoptosis role in leukemic cells. From our study, piperine increased EV production in NB4 and MOLT-4 cells. Moreover, CTSD is involved in EVs from piperine-treated acute leukemia (Pip-EVs). In previous studies, some stimuli could induce or inhibit EV production depending on the mode of action17,50. Additionally, apoptosis is associated with EV production due to an increase in the number of apoptotic bodies or apoptotic EVs51. In NB4 and MOLT-4 cells, Pip-EVs increased CTSD and proapoptotic genes and proteins, including Bax, and caspase-3. Caspase-3 is also required for the release of EVs and their uptake into cells through the cleavage of Bcl-xL52,53. This evidence suggested that once apoptosis is activated by piperine, it results in the release of EVs from cancer cells. Interestingly, natural compounds are unique stimuli that activate EV characteristics and molecules, leading to the transfer of some compounds or biological compounds to target signaling pathways in other cells. In addition, natural compounds could modulate EV cargo proteins and EV function released from cancer cells to improve cancer treatment efficacy54,55. For example, curcumin can alter the levels of bioactive molecules in EVs, such as miR-214 in ovarian cancer-derived EVs and miR-21 in chronic myelogenous leukemia-derived exosomes, consequently suppressing cancer cell growth56. Another example is the traditional Chinese medicine Jianpi Jiedu Recipe, which regulates the loading of ITGBL-1 in EVs and inhibits colorectal cancer metastasis57. According to our previous study, natural compounds including menthol and PG2 peptide induced EV-mediated leukemic cells to enhance apoptosis58,59. From our findings, piperine might influence the release of EVs containing CTSD which were transferred to leukemic cells to promote apoptosis by activating possible molecules such as miRNA or other apoptotic proteins. For the limitation of this study, only two cell lines including NB4, represented as acute promyelocytic leukemia and MOLT-4, represented as T-cell acute lymphoblastic leukemia cells were used in vitro study. For impact of this study and result validation, the exact mechanisms in vivo model and leukemic patient cells should be further investigated.

Conclusion

We demonstrated that piperine inhibits the proliferation of acute leukemic cells by activating the apoptosis signaling pathway via CTSD while having a minimal effect on normal PBMCs. Additionally, piperine activated EV release from acute leukemic cells, increased expression of CTSD and proapoptotic proteins, caspase-3 and Bax. Our findings provide additional insights into the anticancer effect of piperine in acute leukemia, highlighting its potential as an alternative therapeutic strategy.

Materials and methods

Piperine, daunorubicin and pepstatin A

Piperine compound as solid form was purchased from Sigma–Aldrich, Germany. The properties of piperine contained ≥ 97% purity. The stock of piperine was prepared by dissolving lyophilized piperine compound with DMSO and was stored at −4℃. Daunorubicin compound was purchased from TOKU-E, Bellingham, USA. The stock daunorubicin was dissolved in ultrapure water and stored at −4℃. Pepstatin A compound was purchased from Sigma–Aldrich, Germany. The stock of pepstatin A was dissolved in DMSO and stored at −4 ℃.

Leukemic cell culture

NB4 (acute promyelocytic leukemia) and MOLT-4 (T-lymphocytic leukemia) cell lines purchased from Cell Lines Service (Eppelheim, Germany) were used in this study. NB4 cell line, a human acute promyelocytic leukemia cell line, was derived from the marrow of the patient with acute promyelocytic leukemia; M3. MOLT-4 cell line, a T lymphoblast cell line was derived from patient with acute lymphoblastic leukemia (ALL) in relapse. NB4 and MOLT-4 cells were cultured in RPMI-1640 (Gibco™, USA) supplemented with 10% fetal bovine serum (Gibco™, USA) and 1% penicillin‒streptomycin (Gibco™, USA) and incubated in 5% carbon dioxide (CO2) in a 37 °C incubator. The cell culture medium was changed every 2–3 days.

Isolation of peripheral blood mononuclear cells (PBMCs)

The peripheral blood mononuclear isolation protocol was approved by the Mahidol University Central Institutional Review Board (MU-CIRB). Informed consent was obtained from all participants (Ethics approval No. MU-CIRB 2023/125.1204). All methods were performed in accordance with the relevant guidelines and regulations. Whole blood from healthy donors was collected into a 10 ml heparinized tube. Next, whole blood from healthy donors was diluted with PBS and then gently overlaid on Lymphoprep™ solution (STEMCELL™ Technologies, Canada). After centrifugation, a mononuclear cell layer was harvested to study the toxicity of piperine in normal PBMCs via the MTT assay, as described in the MTT assay section.

MTT assay

NB4 cells, MOLT-4 cells, and PBMCs were cultured with 0, 50, 100, or 200 µM piperine (Sigma‒Aldrich, Germany) for 24–48 h. 0 µM of piperine, containing 1% DMSO, was used as a control. After that, 3-[4,5-dimethylthiazol-2-yl]−2,5 diphenyl tetrazolium bromide (MTT) solution (Invitrogen, USA) was added to each well in a 96–well plate to form formazan crystals. After 4 h of incubation with the MTT solution, 10% SDS in 0.01 M HCl was added to stop the reaction and solubilize the formazan crystals. Finally, the absorbance at 570 nm was measured on a microplate reader (BioTek Instruments Inc., USA) to determine the percentage of viable cells. The half maximal inhibitory concentration (IC50) of piperine in NB4 and MOLT-4 cells was calculated from a linear equation established from multiple concentrations of piperine.

Synergistic effect

NB4 and MOLT-4 cells were cultured with the IC50 of piperine combined with 0.2 µM daunorubicin (TOKU-E, Bellingham, WA, USA) for 48 h. NB4 and MOLT-4 cells with 1% DMSO were used as a control. An MTT assay was performed as described previously. The combination index (CI) was calculated using CompuSyn. For interpretation, a CI less than 1, a CI equal to 1, and a CI greater than 1 were considered synergistic, additive, and antagonistic effects, respectively.

Apoptosis assay

NB4 and MOLT-4 cells were cultured with the IC50 of piperine for 48 h. NB4 and MOLT-4 cells with 1% DMSO were used as a control. After harvesting, the cells were washed with PBS. Afterward, Annexin V–conjugated FITC (BioLegend, USA) and propidium iodide (PI) (Sigma‒Aldrich, Germany) were added to stain leukemic cells, which were then incubated for 15 min in the dark. Finally, the percentage of apoptotic cells was determined via a FACSCanto II flow cytometer. A scatter plot of apoptosis was generated via BD FACSDiva™ software.

To confirm CTSD in apoptosis, NB4 and MOLT-4 cells were treated with 10 µM of pepstatin A, a CTSD inhibitor, for 24 h. After that, NB4 and MOLT-4 cells were treated with IC50 of piperine for 48 h. Lastly, NB4 and MOLT-4 cells were stained as mentioned above to determine percentage of apoptotic cells.

Cell cycle assay

NB4 and MOLT-4 cells were cultured with the IC50 of piperine for 48 h. NB4 and MOLT-4 cells with 1% DMSO were used as a control. After harvesting, the cells were washed with PBS. Afterward, the cells were fixed with 70% ethanol in the refrigerator for 30 min. Then, the cells were washed with PBS to remove absolute ethanol. Next, 100 ng/ml RNase A (Roche, Basel, Switzerland) was added to the cells, which were then incubated for 30 min at room temperature. After that, 50 µg/ml propidium iodide (PI) (Sigma‒Aldrich, Germany) was added to the cells, which were incubated for 10 min in cold and dark conditions. A FACSCanto II flow cytometer was used to analyze the cell cycle distribution of NB4 and MOLT-4 cells. The histogram of the cell cycle was established via BD FACSDiva™ Software.

Target protein prediction via bioinformatic tools

The target of piperine was predicted via STITCH (http://stitch.embl.de) and ChEMBL (https://www.ebi.ac.uk/chembl/). Briefly, the keyword “Piperine” was added to STITCH and ChEMBL to generate possible target proteins of piperine. In STITCH, the minimum required interaction score was a high confidence score (0.700), whereas the possible target proteins of piperine from ChEMBL were specified as only homo sapiens and less than or equal to the IC50, EC50, and potency of piperine in NB4 and MOLT-4 cells (150 μm). The proteomic profiles of various cancer cell lines23 were used to predict target proteins of piperine in leukemic EVs. A Venn diagram (https://bioinformatics.psb.ugent.be/webtools/Venn/) was constructed to identify the possible target proteins involved in EVs. A protein–protein interaction (PPI) network between apoptotic proteins, including cathepsin D (CTSD), intracellular cholesterol transporter 1 (NPC1), BID, Bax, and caspase-3 (CASP3), and the possible target proteins involved in EVs was constructed via STRING (https://string-db.org/).

Isolation of extracellular vesicles (EVs)

NB4 and MOLT-4 cells were cultured with the IC50 of piperine for 48 h (Pip-EV). NB4 and MOLT-4 cells with left untreated were used as a control (Con-EV). To isolate EVs, high-speed centrifugation and filtration methods were utilized to harvest EVs. The cells in the supernatant were removed via centrifugation at 1500 rpm for 5 min. The supernatant was filtered with a 0.45 μm vacuum syringe filter and was then centrifuged at 14,000 × g for 45 min at 4 °C. After centrifugation, the supernatant was removed, and the EV pellets were washed with PBS twice via centrifugation at 17,000 × g for 30 min. EV pellets were resuspended in 0.22 μm-filtered PBS, stored at −80 °C for long–term storage, and used to treat leukemic cells in further studies.

EV characterization

Con-EVs and Pip-EVs were observed via transmission electron microscopy (TEM) with negative staining. Afterward, the Con-EV and Pip-EV sizes and particle numbers were quantified via tunable resistive pulse sensing (qNANO Gold, Izon Science Ltd., USA). A 47 nm stretch of Nanopore NP400 (qNANO Gold, Izon Science Ltd., USA) was used to measure EV size and concentration. Con-EVs and Pip-EVs were diluted with measurable electrolyte (ME) buffer. The total particle count was 500 particles for each sample, and 800 Pa pressure was applied to detect the particles. After the EV particles were analyzed, a calibrated particle 400 (CPC400) was used to calibrate each sample for data analysis. Izon data suite software was used to analyze EV size and concentration. The protein concentration of the EVs was determined via a Nanodrop2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) for further experiments. CD9, CD63 (Proteintech, USA), and flotillin-1 (Cell Signaling Technology, USA) antibodies were used to characterize Con-EVs and Pip-EVs via Western blotting as mentioned in “Protein expression measurement by Western blotting”.

Gene expression measurement by real–time quantitative polymerase chain reaction (RT–qPCR)

NB4 and MOLT-4 cells were cultured with the IC50 of piperine for 48 h, 10 µg/ml Con-EVs, or 10 µg/ml Pip-EVs for 24 h. NB4 and MOLT-4 cells with 1% DMSO and Con-EVs were used as a control. GENEzol™ (Geneaid, Taiwan) was used to extract RNA from NB4 and MOLT-4 cells following the manufacturer’s procedure. The RNA concentration was determined via a Nanodrop2000 spectrophotometer (Thermo Scientific, USA). Two hundred to one thousand nanograms of RNA were converted into cDNA via a RevertAid first-strand cDNA synthesis kit (Thermo Scientific, USA). RT–qPCR was performed via Luna® real-time PCR SYBR Green (New England Biolab, Inc., Ipswich, MA, USA) with the designed primers depicted in Table 1. The threshold cycle (Ct) of each sample was quantified via a Bio-Rad CFX96 Touch™ real-time PCR system. The condition for RT-qPCR used in this study was represented in Table 2. The 2−ΔΔCT method was used to calculate the relative mRNA expression. GAPDH, a housekeeping gene, was used to normalize mRNA expression.

Table 1.

Primer sequence used in this study.

Gene name Primer sequence References
CTSD

Forward: 5’-GCAAACTGCTGGACATCGCTTG-3’

Reverse: 5’-GCCATAGTGGATGTCAAACGAGG-3’

 41
Bax

Forward: 5’-CGAGAGGTCTTTTTCCGAGTG-3′

Reverse: 5’-GTGGGCGTCCCAAAGTAGG-3′

 60
Casp3

Forward: 5′-TTCAGAGGGGATCGTTGTAGAAGTC-3′

Reverse: 5′-CAAGCTTGTCGGCATACTGTTTCAG-3′

 60
GAPDH

Forward: 5′-GCACCGTCAAGGCTGAGAA-3′

Reverse: 5′-AGGTCCACCACTGACACGTTG-3′

 60
Open in a new tab

Table 2.

Condition for RT–qPCR.

Order Step Temperature Time
1 Activation 95 ℃ 3 min

2

(40 cycles)

 Denaturation 95 ℃ 10 s
Annealing 60 ℃ 10 s
Extension 72 ℃ 40 s
3 Termination 95 ℃ 20 s
Open in a new tab

Protein expression measurement by Western blotting

NB4 and MOLT-4 cells were cultured with the IC50 of piperine for 48 h, 10 µg/ml Con-EVs, or 10 µg/ml Pip-EVs for 24 h. NB4 and MOLT-4 cells with 1% DMSO and Con-EVs were used as a control. RIPA lysis buffer supplemented with 1% protease inhibitor (Merck Millipore, USA) was used to extract protein lysates from the cells. A Dual-Range BCA Protein Assay Kit (Visual Protein, Taiwan) was used to measure the protein lysate concentration. The protein lysate was separated via SDS–PAGE with a 12% SDS polyacrylamide gel. The proteins on the SDS polyacrylamide gel were transferred to a PVDF membrane, which was blocked with 5% skim milk, 5% BSA, or BlockPRO™ protein-free blocking buffer (Visual Protein, Taiwan). Then, the proteins on the PVDF membrane were incubated with primary antibodies against CTSD, Bax, Caspase-3 (Cell Signaling Technology, USA), and α-tubulin (Proteintech, USA) overnight at 4 °C. Next, the PVDF membrane (GVS Filter Technology, Italy) was incubated with secondary antibodies, including mouse IgG or rabbit IgG conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, USA), depending on the source of the primary antibody. The proteins on the PVDF membrane were detected via an enhanced chemiluminescence system (Bio-Rad, USA). Bio-Rad Image Lab software was used to quantify the band intensity of the proteins. α-Tubulin was used to normalize protein expression.

Statistical analysis

The data from all the experiments are presented as the mean ± standard error of the mean (S.E.M.). All the experiments were performed independently three times (n = 3). Comparisons of two datasets were performed via unpaired t tests. Comparisons of more than three datasets were performed via one-way ANOVA. The results were compared with those of the control or Con-EV treatments. A p value less than 0.05 was considered statistically significant. All graphs and data analyses were performed via GraphPad Prism 8.4.3 (GraphPad Software, USA).

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (3.1MB, pdf)

Acknowledgements

This research project is supported by Mahidol University (Fundamental Fund: fiscal year 2024 by National Science Research and Innovation Fund (NSRF)). The funding source has no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript.

Author contributions

K.C. designed the experiments, conducted the study, performed all experiments, analyzed the data, prepared all figures and wrote the manuscript. W.K designed the experiment, supervised the study and provided resources. S.R. supervised the study and provided resources. D.T. conceptualized, supervised the study, provided resources and approved the manuscript. All authors reviewed the manuscript.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Supplementary Materials

Supplementary Material 1 (3.1MB, pdf)

Data Availability Statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

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