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. 2023 Oct 16;2:100013. doi: 10.1016/j.pscia.2023.100013

Matrine induces V-ATPase-dependent cytoplasmic vacuolation and inhibits the function of the lysosome in leukemia cells

Fanfan Yang a, Wang-jing Zhong a, Jialin Cao a, Junyu Tan a, Bohong Li a, Lingdi Ma b,∗
PMCID: PMC12709986  PMID: 41550167

Abstract

Matrine is the main component extracted from legumes and has extensive anti-cancer effects; however, its molecular mechanism is unclear. In our study, we found that matrine induced vacuolation in leukemia cells is closely related to cell proliferation inhibition. Vacuolization was reversed after matrine removal. The neutral red staining assay indicated that the matrine-induced vacuoles were acidic, and the vacuoles originated mostly from the lysosome or endosome, as observed by transmission electron microscope (TEM) and fluorescence microscopy localization of LAMP-GFP. Furthermore, single-cell RNA sequencing (RNA-seq) demonstrated that the expression of vacuolation- and lysosomal-related genes were up-regulated after matrine treatment, and western blot (WB) and flow cytometry (FCM) analysis confirmed that matrine inhibits intracellular proteolytic enzyme expression and activity, suggesting that matrine may inhibit lysosomal function. In addition, we identified that matrine significantly up-regulated the expression levels of vacuolar ATPase (V-ATPase) subunits in cells, and the V-ATPase inhibitor effectively reversed the occurrence of cell vacuoles, suggesting that V-ATPase plays an important role in matrine-induced vacuoles. The molecular structure of matrine was further analyzed, and the protonation of matrine in lysosomes to activate V-ATPase may be a direct cause of vacuole formation. Our results revealed a new molecular mechanism by which matrine inhibit leukemia cell proliferation.

Keywords: Matrine, Vacuolization, Lysosome, Protonation, V-ATPase

Highlights

  • •

    This study is the first to show that matrine induces vacuolation of leukemia cells.

  • •

    Vacuoles are derived from lysosomes or endosomes and inhibit lysosomal function.

  • •

    The formation of vacuoles is related to protonation.

1. Introduction

Matrine is an alkaloid extracted from traditional Chinese medicines such as Sophora flavescens, Sophora alopecuroides and other legumes. Additionally, it has various other pharmacological effects [1]. Matrine inhibits the growth and proliferation of various tumor cells [[2], [3], [4], [5]]. For example, matrine inhibits chronic myeloid leukemia cell proliferation by down-regulating the ERK/MAPK pathway, mediated by the BCR/ABL fusion gene in chronic myeloid leukemia cells [6]. Matrine induces apoptosis in leukemia cells by inhibiting phosphorylation of the PI3K/Akt/mTOR signaling pathway in leukemia cells [7]. Our team observed that matrine down-regulates HK2 expression by inhibiting c-Myc, thereby inhibiting glycolysis and leukemia cells proliferation [8]. Although the anti-tumor effect of matrine is definite, the mechanism involved remain unclear, and many unknowns remain to be explored.

The lysosome is a digestive organelle, and maintaining its normal function is crucial for maintaining the balance of the intracellular environment. Additionally, it contains a large amount of cathepsin, which is necessary for the cell catabolism and phagocytosis of aging cells [9]. Lysosomal dysfunction leads to cell death of various types [10,11]. After lysosomal damage, cathepsins are released into the cytoplasm to cleaves the BH3-interacting region death agonist BID into a pro-apoptotic tBID fragment, which promoted BAX oligomerization, activates the caspase family, and induces apoptosis [12]. Lysosomal dysfunction leads to cathepsin D release into the cytoplasm, caspase-8 cleavage, and receptor-interacting protein kinases (RIPK-1) activation to induce cell necrosis [13]. Decreased expression and activity of cathepsin affects lysosome digestion and decomposition [14]. A few anti-tumor drugs alter lysosome permeability, leading to the secretion of cathepsins from lysosomes into the cytoplasm, which initiates the lysosomal apoptotic pathway and induces lysosome-dependent cell death [15]. When lysosome function is inhibited, the generation of intracellular iron and lysosomal reactive oxygen species is affected by ferroptosis [16]. Impaired lysosomal function promotes RIPK1 and RIPK3 accumulation, and the necrosis effector protein mixed lineage kinase domain-like protein (MLKL), leading to cell necrosis [17]. Therefore, lysosomes have become potential targets for anti-tumor therapy in recent years.

While studying the effect of matrine on leukemia cells, we observed that matrine could induce vacuolations in human leukemia cells. The vacuoles became more obvious with the prolongation of time and an increase in matrine concentration. The vacuoles were reversed when the matrine was removed. Drug-induced vacuolation promotes cell death [18]. Several factors cause vacuolization. Vacuolization, the formation of vacuoles, is often accompanied by cell death; however, the mechanism underlying cell death remains unclear [19]. Certain lipophilic small- molecule compounds with weak bases enter the lysosome, causing it to absorb water and expand by changing the osmotic pressure to form vacuoles [20]. Small-molecule compounds impede lysosomal maturation by activating ERK kinases, leading to the formation of lysosomal vacuoles and promoting cell death [21]. Akebia saponin E (ASE) induces vacuolization, resulting in liver cancer cells deaths [22].

Our results showed that matrine induces vacuolation in leukemic cells, inhibiting cell proliferation. Matrine-induced vacuoles are derived from lysosomes, endosomes, and inhibit lysosomal function. However, the matrine-induced leukemic cell vacuolation phenomenon has not yet been reported. Both the expression and activity of lysosomal proteolytic enzymes were down-regulated. The occurrence of vacuoles is determined by the chemical structure of the matrine and proton pump of the lysosomal membrane. This study aimed to explore the molecular mechanism of matrine-induced vacuolation in human leukemia cells and its effect on leukemia cells growths. This study further elucidates the pharmacological effects of matrine, provides a meaningful scientific reference for matrine-derived drug development, and provides a new scientific basis for elucidating the molecular mechanism of matrine in leukemia cells.

2. Methods

2.1. Cell lines and reagents

The human leukemia cell lines K562, U937 and HL60 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells are cultured in RPMI 1640 (Corning, USA) with 10% fetal bovine serum (Gibco, USA) at 37°C and 5% CO2. Matrine was obtained from Shanghai Macklin Biochemical Technology Co. Ltd. (Macklin, Shanghai China), and its purity was 98%. A stock solution was prepared in double-distilled water (ddH2O) at 40 mg/mL and stored at 4°C. The different pH values used in this study were configured with a phosphate buffer (pH 2.0; Gibco, USA) and sodium hydroxide (Damao Chemical, China), and the pH was measured using a pH meter (METTLER-TOLEDO-S220K, Switzerland). The experimental techniques used in this study are described in the reagent instruction manual. Ficoll reagent was used to isolate peripheral blood mononuclear cells (PBMCs) from peripheral blood. Informed consent was obtained from each subject, and the study protocol was approved by the Institutional Research Ethics Committee of Huizhou Third People's Hospital.

2.2. Cell proliferation assay

K562, U937 and HL60 cells were treated with matrine, and cell proliferation was analyzed by using the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Japan). The cells were treated with various concentrations of matrine (0.2, 0.4, 0.8, and 1.6 mg/mL) for 24, 48 and 72 h. The cell density is 1.5 × 105/1 × 105/0.75 × 105 per well into 96-well plates for 20, 44, or 68 h, respectively. Then, the CCK-8 reagent was added for 4 h before detection. The optical density (OD) was measured 450 nm using a Microplate Reader (Bio-Rad, USA).

2.3. Western blot and antibodies

Harvested cells were lysed by RIPA lysis buffer (Beyotime, China) for total protein extraction. The protein concentration was measured using a BCA Protein Assay Kit (Beyotime, China). The lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membranes (Immobilon, USA). The membranes were blocked by 5% skimmed milk and incubated overnight with the following primary antibodies: β-Actin (Cat No. 23660-1-AP), CTSB (Cat No. 12216-1-AP), CTSK (Cat No. 11239-1-AP), ATP6AP1 (Cat No. 15305-1-AP), ATP6V1A (Cat No. 17115-1-AP) and ATP6V0D1 (Cat No. 18274-1-AP) (Protein tech, USA), then incubated with HRP-coupled secondary antibodies (Cell Signaling Technology, USA). The protein signal was detected by chemiluminescence reagent (ECL, Beyotime Biotechnology, China) using a high sensitivity chemiluminescence imaging system (Gel Doc™ XR+, Bio-Rad, USA) and quantitatively analyzed by using the Image J software.

2.4. Reverse transcription-polymerase chain reaction (RT-qPCR)

Total RNA was extracted using the TRIzol reagent (Invitrogen, USA) and reverse transcribed into cDNA using the Prime Script RT reagent kit with a gDNA remover (TOYOBO, Japan). cDNA was quantified by real-time PCR using the SYBR Green qPCR kit (Bimake, USA). Relative expression of every gene was calculated by the 2-ΔΔCt method and normalized to β-actin. Primers for all genes were designed using the Primer 3 software and synthesized by Tsingke Biotechnology. Table S1 lists the primer sequences.

2.5. Neutral red staining

Neutral red is a pH indicator that appears red when exposed to acidic conditions and yellow when exposed to alkaline environments. K562 and HL60 cells were treated with matrine for 24 h and U937 cells for 48 h and stained with 1% neutral red (Solarbio, Beijing, China) for 20 min. The supernatant was discarded after centrifugation, 1 mL of 1 × PBS was added, and the cells were mixed. Take 20 μL on a glass slide and observe the staining of cells using an optical microscope (Olympus, Japan).

2.6. Transmission Electron Microscope (TEM) analysis

Control and treatment groups of K562 cells were fixed with 2.5% glutaraldehyde, 1 M phosphate buffer (pH7.4) washed three times, 15 min each time; 1% osmic acid 0.1 M phosphate buffer (pH7.4) room temperature (20°C) fixed for 2 h; 0.1 M phosphate buffer (pH7.4) washed three times, 15 min each time; 50%–70%–80%–90%–95%–100%–100% alcohol is dehydration, 15 min each time; Acetone: 812 packets Embedding agent = 1:1 mixed solution are infiltrated overnight; the sample was polymerized at 60°C for 48 h. The prepared sample was cut 60–80 nm ultrathin and stained with 2% uranyl acetate saturated aqueous solution and lead citrate for 15 min to dry overnight at room temperature, and then observed under a transmission electron microscope (FEI Tecnai G20 TWIN,USA) and photographed.

2.7. RNA-sequencing experiment and analysis

RNA was extracted using a TRIzol reagent (Invitrogen, USA). After extraction, the total RNA from each sample was quantified and qualified to inspect the RNA integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Next-generation sequencing library preparation was perfromed per the manufacturer's instruction. Poly(A) mRNA was isolated using a Poly (A) mRNA Magnetic Isolation Module or rRNA Removal Kit. Libraries with different indices were multiplexed and loaded onto an Illumina HiSeq instrument per the manufacturer's instructions (Illumina, USA). Sequencing was carried out using a 2 × 150 bp paired-end (PE) configuration. The sequences were processed and analyzed using GENEWIZ. RNA-seq reads were mapped to the human genome reference assembly (hg38) using Hisat2 (v2.0.1). HTSeq (v0.6.1) was used to estimate the gene and isoform expression levels. Significant differentially expressed genes were identified as those with an adjusted p-value above the threshold (| log2 (fold change |≥1.8, Padj≤0.05 and FPKM≥1 using DESeq2 software), between the two groups were analyzed using Top GO_CC. Gene Set Enrichment Analysis (GSEA) was used to analyze the significant differentially expressed genes (DEGs) using the Cluster Profiler R package.

2.8. Fluorescence microscopy analysis

To confirm the specific location of the vacuoles, a lysosomal membrane marker fused with a green fluorescent protein (LAMP1-eGFP) was cloned into a lentiviral vector (pRRLSIN-cPPT-SFFV-MCS-SV40-puromycin) in leukemia cells. Shanghai GeneChem performed both vector construction and virus packaging. Leukemic cells were infected with the virus at an MOI of 30 to construct stable live cell lines. After matrine treatment, the green fluorescence distribution in the cells was observed using a Leica fluorescence microscope (DMI8), which emits green fluorescence excited by blue light.

2.9. DQ-green BSA assay

DQ Green BSA is composed of the fluorescent dye BODIPY and bovine serum albumin derivatives. BODIPY exhibit self-quenching properties. These two substances are coupled and do not emit light. When the protease digests and hydrolyzes BSA protein, the protein fragments produced emit bright fluorescence; therefore, DQ Green BSA monitors BSA proteolysis, and the stronger the hydrolase activity, the more fluorophores are released. General lysosomal protease activity was measured by DQ green BSA (Thermo Scientific, USA), 1 × 106/mL cells were incubated with 10 μg/mL DQ green BSA for 3 h at 37°C, 5% CO2, 0.8 mg/mL matrine was added and cultured for 24 h, and washed 3 times with PBS (Gibco, USA) for 5 min each, and fluorescence intensity was assessed using flow cytometry (BD FACS Canto, USA) [23].

2.10. Statistical analysis

Data were analyzed using GraphPad Prism 7 software and SPSS 25.0. The quantitative data were represented as means ± standard deviation. In this study, a t-test was applied to compare the means of the two groups. Differences were considered statistically significant at a p-value of <0.05.

3. Results

3.1. Matrine induces vacuolation in human leukemia cells

To understand the effect of matrine on leukemia cells morphology, cells were treated with different matrine concentrations, and cell morphology was observed using an optical microscope. Matrine induces vacuoles in human leukemia cells obviously. The higher the matrine concentration, the larger the vacuole volume (Fig. 1a). The calculated percentages of vacuolated cells showed that the higher the matrine concentration, the more vacuoles the cells contained (Fig. 1b). Peripheral blood mononuclear cells were isolated from healthy individuals and patients with leukemia and cultured. When the cells were stabilized, PBMCs were treated with matrine for 48 h, and cell activity was detected. We observed that the number of vacuoles in normal cells was lower than that in tumor cells, and that matrine inhibited leukemia cell proliferation; however, it did not affect the activity of normal PBMCs, indicating that matrine was selective for tumor cell growth and did not affect normal cells (Fig. S1). K562 cells were treated with 0.8 mg/mL matrine for 24 h, and TEM confirmed the presence of vacuoles (Fig. 1c). It is suggested that matrine induces vacuoles formation in different leukemia cells.

Fig. 1.

Fig. 1

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Matrine induces vacuolation in human leukemia cells.a K562 and HL60 cells were treated with different concentrations (0.4 mg/mL, 0.8 mg/mL, and 1.6 mg/mL) of matrine for 24 h, and U937 cells were treated for 48 h. The cell morphology was observed using an optical microscope, bar = 20 μm. b Calculated percentages of the vacuolated cells = the number of cells with vacuoles in 15 fields/the total number of cells in 15 fields × 100%. c K562 cells were treated with 0.8 mg/mL matrine for 24 h, and cell vacuoles were observed using TEM, bar = 10 μm ∗p < 0.05, ∗∗p < 0.01. Red arrows indicate vacuoles.

3.2. Vacuolation was closely related to cell proliferation

To further explore the relationship between vacuoles and cell proliferation, the cells were treated with different matrine concentrations for 24 h, 48 h, and 72 h, respectively. The results showed that matrine effectively inhibited human leukemia cells proliferation in a dose- and time-dependent manner (Fig. 2a). K562 cells were treated with 0.8 mg/mL matrine for 24 h, vacuoles occurred in the cells; matrine was removed; the cells were cultured for 24 h; he vacuoles disappeared, and cell viability was restored (Fig. 2b and c). K562 cells were treated with matrine for different periods, vacuoles were observed, and cell proliferation was measured. The higher the concentration and the longer the matrine treatment duration, the more vacuoles appeared in the cell and the lower the cell viability, until finally the cells burst and died (Fig. 2d). These data were in accordance with cell viability the assessment, indicating that vacuoles play an important role in inhibiting human leukemia cell proliferation.

Fig. 2.

Fig. 2

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Cell viability decreases with increasing vacuolization.a 0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL and 1.6 mg/mL of matrine were used to treated leukemia cell lines K562, HL60 and U937 cells for 24 h, 48 h and 72 h, and the cell proliferation activity was detected by CCK8 assay. b,c K562 cells were treated with 0.8 mg/mL matrine for 48 h, compared with 0.8 mg/mL matrine for 24 h, and the drug was removed to culture for 24 h (MAT + -). d The cell morphology was observed using an optical microscope, and the cell proliferation activity was detected by CCK8 assay, bar = 20 μm. K562 cells were treated with 0.8 mg/mL matrine for different time (12 h, 24 h, 36 h, 48 h, 60 h, 72 h) to observe the changes of vacuoles, and cell proliferation activity was detected by CCK8 assay, bar = 20 μm ∗p < 0.05, ∗∗p < 0.01. Red arrows indicate vacuoles.

3.3. Matrine-induced vacuoles localize to lysosomes

A neutral red dye was used to determine the origin of the vacuole and whether it was acidic or alkaline. Nuclei were stained in the control group, and vacuoles were stained red in the matrine-treated group (Fig. 3a), indicating that matrine-induced vacuoles are acidic. The lysosomal membrane marker LAMP1-eGFP was used to label lysosomes. The results showed that fluorescence was punctately distributed in the cells before matrine treatment, and fluorescence appeared in the periphery of the vacuoles after matrine treatment (Fig. 3b), indicating that the vacuole was derived from the lysosome or endosome. Subsequently, we observed the ultrastructure of cell vacuoles using TEM (Fig. 3c). As the matrine treatment duration was prolonged, the vacuole volume increased, and the vacuoles contained contents that were not completely decomposed and digested, similar to the digestive organ-lysosome. These data indicated that matrine-induced vacuoles might originate from lysosomes or endosomes.

Fig. 3.

Fig. 3

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Matrine-induced vacuoles originate from lysosomes. a K562 and HL60 cells were treated with 0.8 mg/mL matrine for 48 h, and 1.6 mg/mL matrine was treated with U937 cells for 24 h, stained with 1% neutral red for 20min, and the vacuoles staining was observed using an optical microscope, bar = 10 μm. b K562 and HL60 cells overexpressing LAMP1-eGFP were treated with 0.8 mg/mL matrine for 24 h, and 1.6 mg/mL matrine was treated with U937 cells overexpressing LAMP1-eGFP for 48 h. The fluorescence distribution was observed using a fluorescence, icroscope, bar = 20 μm. c K562 cells were treated with matrine for 12 h, 24 h, and 36 h, and vacuoles were observed by TEM, bar = 10 μm ( × 1.5 K), bar = 1 μm ( × 8.0 K) (c). V: vacuoles, N: nucleus, Er: endoplasmic reticulum, Mt: mitochondria, AV: autophagosome.

3.4. Matrine up-regulates vacuolation and lysosomal-related genes

To understand the changes in gene expression in leukemia cells after matrine treatment, K562, HL60 and U937 cells were treated with 0.8 mg/mL matrine for 24 h, and total cellular RNA was extracted for RNA-seq. In matrine-treated leukemia cells, 185 genes were significantly up-regulated and 241 genes were significantly down-regulated by cell component analysis. These up-regulated genes are related to vacuolization and lysosomal processes (Fig. 4a). In addition, we analyzed GO_BP and GO_MF. Regarding its biological functions, matrine promotes sterol biosynthesis and inhibits ribosome biogenesis. Matrine upregulated kinase inhibitor activity and inhibited RNA binding (Fig. S2). Eighteen vacuolation-related genes and 15 lysosome-related genes highly overlapped (Fig. 4b). The upregulation of 15 overlapping genes in matrine-treated human leukemia cells is represented by a heat map (Fig. 4c). Matrine upregulated the mRNA expression levels of vacuolation-related genes (marked in red in Fig. 4c), which was consistent with the RNA-seq results (Fig. 4d). This result indicates that matrine-induced vacuolation may be related to lysosomes, and that these overlapping genes may cause of matrine-induced vacuolation.

Fig. 4.

Fig. 4

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Matrine upregulated vacuolation and lysosome-related genes, which highly overlapped the two. a K562, HL60 and U937 cells were treated with 0.8 mg/mL matrine for 24 h, then RNA-seq was performed. Differential genes were screened according to |log2|≥1.8, Padj≤0.05, and FPKM≥1, and the up-regulated genes and down-regulated genes were enriched by GO_CC. b Venn diagram showing the intersection of 18 vacuolation-related genes and 15 lysosome-related genes in human leukemia cells treated with matrine. c Drawing a heat map representing the expression levels of vacuolation-related genes in matrine-treated human leukemia cells. d RT-qPCR confirmed that matrine up-regulated vacuolation-related genes in RNA-seq. ∗p < 0.05, ∗∗p < 0.01.

3.5. Matrine inhibited lysosomal function

Furthermore, to confirm that matrine affects lysosome function, we conducted the following experiment and detected the expression levels of lysosomal proteolytic enzymes, cathepsin B (CTSB) and cathepsin K (CTSK), in human leukemia cells. RT-qPCR verified the CTSB and CTSK expressions after matrine treatment for 24 h in K562, HL60 and U937 cells, and the results confirmed that matrine up-regulated their mRNA levels (Fig. 5a). The CTSB and CTSK protein levels significantly decreased, indicating that matrine affected the lysosomal proteolytic enzyme expression (Fig. 5b). We then treated K562 cells with 0.8 mg/mL matrine for 24 h, and detected the total proteolytic enzyme activity of lysosomes. The fluorescence intensity of DQ-Green BSA was significantly weakened, and the total proteolytic enzyme activity of the lysosomes decreased (Fig. 5c). The data showed that matrine inhibited proteolytic enzyme expression and activity, thereby inhibiting lysosomal function.

Fig. 5.

Fig. 5

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Matrine inhibits lysosomal function. a K562, HL60 and U937 cells were treated with 0.8 mg/mL matrine for 24 h, and the CTSB, CTSK mRNA level was detected by RT-qPCR. b K562 and HL60 cells were treated with 0.8 mg/mL matrine for 24 h, and 1.6 mg/mL matrine was treated with U937 cells for 24 h. The protein expression levels of CTSB and CTSK were detected by western blot. c K562 cells were treated with 0.8 mg/mL matrine for 24 h, and the intracellular DQ-Green BSA fluorescence intensity was detected by FACS, ∗∗p < 0.01.

3.6. Matrine activates the lysosomal proton pump V-ATPase

To explore the molecular mechanism of matrine-induced cytoplasmic vacuolation in leukemia, we searched for information ona molecule—vacuolar ATPase (V-ATPase), as a proton pump in lysosomes [24,25] which was originally observed in yeast and plant vacuoles. Moreover, it is a complex composed of V1 and V0 proteins. V1 is located on the membrane surface, V0 is located in the membrane, and both V1 and V0 have different subunits [26]. V-ATPase maintains the acidic environment of lysosomes and activates of V-ATPase-induced vacuoles [27,28]. BafA1, a V-ATPase inhibitor, is a macrolide antibiotic isolated from Streptomyces sp. In a previous study, BafA1 impaired the formation of vesicular intermediates between early and late endosomes [29]. As another V-ATPase inhibitor, concanamycin A (CMA) is a macrolide antibiotic that acts by binding to the V0 domain of the proton pump V-ATPase [30]. V-ATPase inhibitors inhibit V-ATPase activation and prevent the pumping of hydrogen into lysosomes. By analyzing the V-ATPase mRNA expression levels of subunits using RNA-seq, we observed that matrine upregulated the gene expression levels of V-ATPase subunits (Fig. 6a). RT-qPCR verified the expression of matrine in the V-ATPase subunit and the results confirmed that matrine upregulated V-ATPase subunit mRNA levels (Fig. 6b). HL60 cells were treated with 0.8 mg/mL matrine for 24 h, and matrine upregulated the protein expression level of V-ATPase subunit (Fig. 6c). Subsequently, leukemia cells were treated with the V-ATPase inhibitors CMA or BafA1 and matrine, and few vacuoles were observed, especially in K562 and HL60 cells (Fig. 6d). The calculated percentages of vacuolated cells are shown in (Fig. 6e). In leukemia cells treated with the V-ATPase inhibitors CMA or BafA1, cell morphology was immutable. V-ATPase inhibitors effectively reduced vacuole formation following matrine treatment. These results suggested that matrine-induced vacuolization was related to V-ATPase activation.

Fig. 6.

Fig. 6

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Matrineup-regulatedtheV-ATPaseexpression level. a RNA-seq data analysis of V-ATPase subunit mRNA expression level; b K562, HL60 and U937 cells were treated with 0.8 mg/mL matrine for 24 h, and the V-ATPase subunit mRNA level was detected by RT-qPCR. c HL60 cells were treated with 0.8 mg/mL matrine for 24 h, and the protein levels of V-ATPase subunit was detected by western blot. d K562 and HL60 cells were treated with 0.8 mg/mL matrine for 24 h and U937 cells were treated with 1.6 mg/mL matrine for 48 h, combined with CMA (1 nM for K562, 0.4 nM for HL60, 0.4 nM for U937) or BafA1 (K562, HL60 for 10 nM, U937 for 20 nM), cell morphology was observed by optical microscope, bar = 20 μm. e Calculated percentages of the vacuolated cells. ∗p < 0.05, ∗∗p < 0.01. Red arrows indicate vacuoles. FC: Fold Change.

3.7. Protonation of matrine may be the direct cause of vacuolation

Furthermore, to investigate the effect of the chemical structure of matrine on its activity, we analyzed the structure of matrine and its analogs, which showed that oxymatrine adds an oxygen atom to the nitrogen atom at position 1. In contrast, matrine and sophoridine do not have this structure (Fig. 7a), which may prevent nitrogen protonation at position 1 of oxymatrine. Our results demonstrated that matrine and sophoridine induced cell vacuolation; however, oxymatrine did not (Fig. 7b and c), suggesting that the reason for vacuolation may be nitrogen protonation at position 1 of matrine and its analogs. To further explore the effect of matrine-induced cell vacuolization and cell proliferation under different pH conditions, HL60 cells were treated with matrine at different pHs for 24 h. Matrine's ability to induce cell vacuoles was weakened in pH 2.0 and pH 9.0 solvents. However, not in pH 4.0 and pH 5.0 solvents (Fig. 7d and e), while in pH 2.0 solvents, matrine's ability to inhibit cell proliferation was significantly weakened (Fig. 7f). This suggests that the ability of matrine to induce vacuoles formation may be related to pH and that matrine may induce vacuole formation by protonation. Under acidic conditions, matrine molecules are protonated outside the lysosomes, preventing them from entering the lysosomes and changing the osmotic pressure.

Fig. 7.

Fig. 7

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Matrine-induced vacuolation is related to the amine group at position 1.a Molecular structural formula of matrine, sophoridine and oxymatrine. b K562 and HL60 cells were treated with 0.8 mg/mL matrine analogs for 24 h, and 1.6 mg/mL matrine analogs were treated with U937 cells for 48 h. The cell morphology was observed by optical microscope, bar = 20 μm. c Calculated percentages of the vacuolated cells. The p-value is compared to control group. d HL60 cells were treated with 0.8 mg/mL matrine at different pH (pH 2.0, pH 4.0, pH 5.0 and pH 9.0) for 24 h, and the changes of cell vacuoles were observed by optical microscope, bar = 20 μm. e Calculated percentages of the vacuolated cells. Except for special lines, the p-value is compared to control group. f HL60 cells were treated with 0.8 mg/mL matrine at different pH for 48 h, and cell proliferation activity was detected by CCK8 assay. ∗p < 0.05, ∗∗p < 0.01. The red circle refers to the nitrogen atom of matrine position 1. Red arrows indicate vacuoles.

4. Discussion

Matrine induced vacuoles in human leukemia cells are closely associated with cell proliferation. Increasing evidence is available on the cytoplasmic vacuolization of human tumor cells by small-molecule drugs, which has a propulsive effect on inducing cell death [31,32]. Rac1 activates Arf6-GAP and GIT-1 proteins to impair the function of the Arf6-GTP protein, leading to the inactivation of Arf6 protein to induce vacuolation [33]. Doxycycline induces vacuolation in the tumor cell lines DU145, MDA-MB-468 and A549 by activateing H-Ras and increasing Rac1 expression; downregulation of Rac1 reverses cytoplasmic vacuolation and cell death [34]. In glioblastoma cells, the mitochondrial membrane potential and ATP levels are increased by up-regulation of H-Ras. These changes disrupt cellular metabolic function and prevent ATP-mediated fusion of late endosomes and lysosomes, eventually, the continuous accumulation of vacuoles destroys cell integrity and leads to cell death [35]. Similarly, matrine inhibited cell proliferation by inducing vacuolation in leukemia cells.

Following overexpression of the lysosome labeled molecule LAMP1 fused with eGFP, a large amount of green fluorescence was observed at the cell vacuolar edge after matrine treatment by fluorescence microscopy, indicating that the vacuoles originated from lysosomes, and not from mitochondria or Golgi bodies. Since LAMP1 can be distributed in late endosomes, vacuoles can occur in late endosomes, which require labeling with the late endosomal marker Rab7 for identification. Autophagosomes are spherical structures with double or multilayer membranes, containing cytoplasmic components with a diameter of 100–900 nm [36]. Research has reported matrine-induced vacuoles in hepatoma G2 cells, which were shown to be autophagosomes [37], in our study, matrine-induced vacuolation of leukemia cells was derived from lysosomes or endosomes, and matrine-induced autophagosomes were shown in Fig. 3c, which were inconsistent with the vacuoles. Previous studies [38] have confirmed that matrine enters the lysosomes of tumor cells and increases the pH of the lysosome.

In addition, Borkowska reported that the TMA (5.3 ± 0.7 nm gold NPs18, 29, 32 functionalized with positively charged N, N, N-trimethyl (11-mercaptoundecyl) ammonium chloride) and MUA (negatively-charged 11-mer-captoundecanoic acid) ligand mixtures were co-cultured with tumor cells to target the lysosome. Nanoparticles aggregate in the lysosome to increase osmotic pressure, which makes the lysosome absorb water and expand. Finally, the lysosome ruptures and cell death: the nanoparticles TMA: MUA (4:1) kill tumor cells. Cationic ligands of nanoparticles are cytotoxic to both cancer and healthy cells, and anionic ligands of nanoparticles are not well attached to the negatively charged membrane because it is difficult to internalize and lose their effect on tumor destruction [39]. This result is similar to that of our study: matrine may be retained after entering the lysosome, inducing osmotic changes and lysosomal swelling to form vacuoles, and vacuole production also inhibits lysosomal function. With further research, lysosomes have become important targets for anti-tumor therapy, and targeting lysosomes kill tumor cells is necessary [40,41]. Combined with these research results, we believe that the occurrence of vacuoles leads to the downregulation of lysosomal proteolytic and autophagy related protein accumulation, which leads to lysosomal dysfunction and affects the proliferative activity of human leukemia cells.

During vacuolization, V-ATPase acts as a hydrogen donor in the acidic environment of lysosomes, pumping hydrogen ions from the lysosomal membrane into the lysosome, maintaining a high hydrogen ion concentration in the lysosome, and maintaining a significant difference in the hydrogen ion concentration inside and outside the lysosome, thereby balancing the acidic environment in the lysosome. Our study observed that sophoridine significantly induced vacuolation among the matrine structural analogs, whereas oxymatrine did not. The matrine molecular structure analysis revealed that it and sophoridine contain free amine groups that can bind hydrogen ions, whereas oxymatrine does not. In addition, matrine-induced cell vacuolization can be affected by changing the pH of matrine, suggesting that cellular vacuolation is related to matrine protonation. In the early years, Christian de Duve proposed a mechanism for the accumulation of lipophilic amine small molecules in the acidic environment of lysosomes: amine-containing weakly basic drugs and uncharged drugs enter cells by simple diffusion; they enter the cytoplasm and are transported into organelles. When these molecules diffuse into acidic organelles and protonate with hydrogen ions, they are charged and trapped in them. The higher the osmotic pressure in the organelles, the more the organelles swell, and vacuoles were observed using an optical microscope. Prolonged drug treatment and elevated drug concentrations lead to the gradual enlargement of vacuoles, ultimately resulting in their rupture and death [42]. Therefore, we believe that protonation of matrine may be an important mechanism for the induction of vacuoles, and lysosomal function inhibition by matrine-induced vacuolization is one of the mechanisms by which matrine exerts its anti-leukemic effects.

Another reason for vacuolation is related to the content of PI (3,5) P2, a phospholipid signaling molecule that maintains homeostasis of the vesicle system and plays a critical role in the vesicle transport pathway of eukaryotic cells. In yeast, PI (3,5) P2 levels are determined by the pathway balance involved in the synthesis of Vac14. Vac7 activats Fab1 kinase and dephosphorizes of PI (3,5) P2 to PI3P (Fig. 4) [43]. The relationship between intracellular PI (3,5) P2 levels and vacuoles can be described as follows. First, the PI (3,5) P2 signal is an important regulatory factor of vacuole/lysosome morphology, and the lack of intracellular PIKfyve and the reduction in PI (3,5) P2 content lead to the rapid expansion of lysosomes to form vacuoles [44,45]. Another reason is that reduced PI (3,5) P2 synthesis leads to a large accumulation of multiple cations (with the highest potassium ions concentration) during transport, forming vacuoles [46]. Therefore, PI (3,5) P2 in cells has an important effect on vacuoles. To investigate whether matrine induced vacuolation of human leukemia cells is related to PI (3,5) P2, K562 cells were treated with 0.8 mg/mL matrine for 24 h, and FACS detected the fluorescence intensity of PI (3,5) P2 in the cells. The results demonstrated that the fluorescence intensity in the cells did not change after matrine treatment; in other words, the PI (3, 5) P2 content did not change with matrine treatment (Fig. S3), suggesting that matrine-induced vacuoles are independent of PI (3, 5) P2.

Matrine possesses extensive anti-leukemc effect, is a traditional Chinese medicine with low toxicity, rarely destroys normal cells, and has good clinical application prospects for treating patients with leukemia. However, in most studies, the anti-leukemic effect of matrine is mainly demonstrated in in vitro, and there are few animal experiments or clinical experiments. Due to the special structure of matrine, finding its targets of action by labeling matrine with biotin or fluorophores is a major obstacle. However, the short half-life and high concentration of matrine limit its clinical application. Researchers need to make more efforts to label matrine, synthesize new matrine derivatives, and comprehensively elucidate the anti-leukemia effects and molecular mechanisms of matrine by combining in vitro and in vivo studies with various experimental techniques. Based on current research results, future studies should prioritize matrine as a crucial molecular mechanism underlying the formation of cytoplasmic vacuoles. This focus will provide a deeper understanding of matrine's pharmacological properties and facilitate the development of diverse matrine-based therapies. By targeting lysosomes, these therapies hold promise for the treatment of cancer cells, offering valuable insights for the advancement of Chinese medicine and traditional medicine as a whole.

5. Conclusion

In summary, matrine enters the lysosome, lysosome; the acidic environment of the lysosome protonates matrine in the lysosome, increasing the osmotic pressure in the lysosome; and local vacuoles absorb water and swell, leading to cytoplasmic vacuolization. In contrast, matrine is protonated in the lysosome to consume hydrogen ions, and the lysosome activates V-ATPase to maintain its acidic environment. In this study, we observed for the first time that matrine induced vacuolation of leukemia cells and that vacuolation originated from lysosomes and inhibited lysosome function, which provides an important reference value for matrine-targeting lysosome therapy of leukemia cells with strong innovation.

Date availability

The accession number for the RNA-Seq of the three leukemia cell lines treated with matrine reported in this article were deposited in the Gene Expression Omnibus (GEO: GSE201309).

Author contributions

F.F.Y. designed the study, performed and analyzed all experiments, and wrote the manuscript. W.-J.Z., J.L.C., J.Y.T., and B·H.L. performed the experiments and interpreted the data. L.D.M. supervised the project, designed the study, and wrote the manuscript. All the authors have read and approved the final manuscript.

Funding

This work was supported by the National Nature Science Foundation of China (81673644), the Research Projects of Guangdong Provincial Bureau of Traditional Chinese Medicine (20181261), and the Science and Technology Plan (Medical and Health) Project of Huizhou (2018Y158).

Ethics approval

Not applicable.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgments

Not applicable.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.pscia.2023.100013.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

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