Matrine-Induced Apoptosis in Leukemia U937 Cells: Involvement of Caspases Activation and MAPK-Independent Pathways

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Reagents and cell culture
• Annexin V-FITC/propidium iodine FACS
• DNA laddering analysis
• Western blot analyses
• Cytochrome C release assay
• Cytotoxicity assay
• Results
• Discussion
• Acknowledgements
• References

Abstract

It is reported that matrine, one of the major effective compounds isolated from the root of Sophora flavescens Ait., has anti-leukemia activity. In this study, we find that the treatment of leukemia U937 cells with matrine results in induction of apoptosis. Analysis of the mechanism underling this apoptotic event showed activation of caspases-9, -3, and -7, and release of cytochrome C from mitochondria to cytosol, and cleavage of poly(ADP-ribose) polymerase. Matrine did not alter the level of bcl-2 and bcl-xL as well as bax. In addition, no correlation was found between matrine administration and activation of the three major MAPK subfamilies (Erk1/2, p38, JNK/SAPK). The results indicate that matrine induces apoptosis in U937 cells via a cytochrome C-triggered caspase activation pathway.

Abbreviations

MAPK:mitogen activated protein kinase

JUN/SAPKs:c-Jun N-terminal kinases/stress-activated protein
kinases

Erk1/2:extracellular signal-related kinases

Introduction

Sophora flavescens Ait. is a leguminous plant growing in China, Japan and some European countries. Its dry root (SF) is commonly used in Traditional Chinese Medicine to treat a range of diseases, including cancer, viral hepatitis, cardiac arrhythmia, and skin diseases [1]. The alkaloid matrine (Fig. [1]) is one of the major bioactive components in SF. Currently, matrine injection has been utilized to treat hepatitis patients in China [2]. Previous studies indicate that matrine is an effective agent in growth inhibition and induction of cell differentiation in leukemia K562 cells [3]. However, the molecular mechanism underlying the cytotoxicity of matrine on leukemia cells is largely unknown.

Apoptosis, or programmed cell death, plays a pivotal role in controlling development and homeostasis in all multicellular organisms. Deregulation of apoptosis has been recognized as one of the fundamental aspects of tumor biology [4]. Many chemotherapeutic agents appear to eliminate tumor cells by activating key elements of apoptosis [5]. The molecular mechanism underlying apoptosis generally involves the activation of caspase (cysteine aspartase) family members [6]. Caspases are commonly divided into two groups, the upstream initiator caspases including caspases -2, -8, -9 and -10 and the downstream effector caspases including caspases -3, -6 and -7 [7], [8]. Stimulation by chemotherapeutic agents leads to processing and activation of initiator caspases, which subsequently transmit the signal to downstream effector caspases.

Signal transduction pathways participate in the regulation of apoptosis induction by chemotherapeutic agents [9]. The mitogen-activated protein kinases (MAPK) consisting of three major subfamilies, c-Jun N-terminal kinases/stress-activated protein kinases (JUN/SAPKs), extracellular signal-related kinases (P42/44 MAPK; Erk1/2), and P38 MAPK, mediate signal pathways involved in cell survival and death. The role of MAPK in apoptosis is cell type- and stimulus-dependent. In some cases, the activation of the SAPK and P38 MAPK cascades promotes apoptosis, whereas the activation of Erk1/2 exerts a cytoprotective effect [10], [11].

The purpose of this study is to investigate the cytotoxic effects of matrine on U937 cells and its underlying mechanisms. Based on the findings that matrine indeed induced apoptosis, we further examined activation of caspase-3, -9, and -7 and release of cytochrome C, and level of bcl-2 and bcl-xL as well as bax proteins. In addition, we also investigated the influence of matrine on activation of MAPK, one of the signal transduction pathways.

Materials and Methods

Reagents and cell culture

Matrine was kindly provided by Ningxia Yanchi Pharmaceutical Plant, P. R. China, and its purity was > 99 % by high-performance liquid chromatography (HPLC). Matrine was prepared as a 10 mg/mL stock solution in sterile H₂O. U937 cells were cultured in RPMI 1640 supplemented with penicillin, streptomycin, and 10 % FCS. They were maintained in a 37 °C, 5 % CO₂, fully humidified incubator, passed twice weekly. Logarithmically growing cells were collected at a concentration of 1 × 10⁵ cells/mL, to which were added the designated drugs, and the cells were placed back into the incubator for another 24 h or 48 h. At the end of the incubation period, cells were transferred to sterile centrifuge tubes, pelleted by centrifugation, and prepared for analyses as described below.

Annexin V-FITC/propidium iodine FACS

Apoptosis of each sample was determined by flow cytometry using a commercially available (Bender MedSystems Inc., USA) annexin V-FITC/propidium iodine apoptosis detection kit. After drug treatment, cells were washed twice in ice-cold PBS and resuspended in 250 μL of binding buffer at 1 × 10⁵ cells/mL. 100 μL of suspension were taken and incubated with 5 μL of annexin V/FITC and 10 μL of propidium iodine (20 μg/mL) in the dark for 15 min at room temperature. Finally, 400 μL of PBS were added to each sample and samples were analyzed by flow cytometry and evaluated based on the percentage of cells staining low or high for annexin V (apoptotic cells).

DNA laddering analysis

Apart from FACS, an apoptotic DNA-ladder kit (Boehringer Mannheim, Gmbh, Germany) was used to determine the presence of DNA laddering in apoptosis induction. Briefly, the cell samples were collected in lysis buffer and the genomic DNA was column-isolated by centrifugation in an elution buffer. The genomic DNA was subjected to electrophoresis on a 1 % agarose gel containing ethidium bromide and visualized by UV.

Western blot analyses

To obtain the changes of protein expression in apoptosis induction by matrine, a modified method of Western blot analyses as previously described was used [12]. Briefly, collected cells were lysed immediately in buffer [1 % Triton X-100, 150 mM NaCl, 25 mM Tris-HCl pH7.2, 0.5 mM EDTA, 0.5 mM Na₃VO₄] supplemented with a protease inhibitor cocktail (Roche Molecular Biochemicals, Germany). Protein concentration was determined (Micro BCA kit; Beyotime Biotechnology, P.R. China). Equal amounts of protein (60 μg) were boiled for 5 min, separated by SDS-PAGE, and electroblotted to a nitrocellulose membrane. After blocking, the blots were incubated with an appropriate dilution of specific antisera or monoclonal antibodies (PARP; caspase -3, -9, -7; bcl-2,bcl-xL,bax; phospho-Erk1/2, phospho-P38, phospho-JNK/SAPK; Cell Signaling Technology, USA) for 1 h at room temperature. Blots were washed three times and then incubated with a 1 : 2000 dilution of horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, USA) for 1 h at room temperature. Blots were again washed three times and then developed using a chemiluminescence assay. Blots for β-actin (Cell Signaling Technology, USA) were used as a loading control. The optical densities for cytochrome C and bcl-2 family proteins were measured with the Scion Image Analysis System (Scion; Frederick, MD, USA) and standardized using the densities for β-actin.

Cytochrome C release assay

Assay kits for cytochrome C release (Calbiochem, Germany) were used to assess the release of cytochrome C from mitochondria to cytosol. Briefly, cell samples were harvested and washed once with ice-cold phosphate-buffered saline (PBS) by centrifugation at 600 × g for 5 min at 4 °C. Cell pellets were resuspended in cytosol extraction buffer, incubated on ice for 10 minutes and homogenized in an ice-cold tissue grinder for 30 passes. The homogenate was centrifuged at 700 × g for 10 minutes at 4 °C and the supernatant was centrifuged at 10,000 × g for 30 minutes at 4 °C. The supernatant was harvested as the cytosolic fraction. The pellets were resuspended in mitochondrial extraction buffer and saved as mitochondrial fraction. 10 μg of each cytosolic and mitochondrial fraction were loaded on a 12 % SDS-PAGE for the standard procedure of Western blotting. The optical densities of the bands for cytochome C were measured with the Scion Image Analysis System (Scion; Frederick, MD, USA).

Cytotoxicity assay

After exposure of the cells in 96-well dishes to matrine for the indicated intervals, cell viabilities were assessed by the 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium (MTT) assay [13]. Briefly, U937 cells were collected and resuspended in RPMI 1640 medium at 1 × 10⁵ cells/mL. 90 μL aliquots were added to each well of 96-well flat-microtiter plates, followed by addition of 10 μL of ddH₂O containing concentrations of the drugs. Three replicate wells were used for each data point in the experiments. After incubation for the indicated intervals, 10 μL of MTT 9 (Sigma, USA) solution (5 mg/mL in ddH₂O) were added to each well and plates were then incubated for 4 h at 37 °C. Intracellular formazan crystals were dissolved by addition of 100 μL isopropyl alcohol with 0.04 N HCl to each well, until the solution turned purple and absorbance analyzed in an enzyme-linked immunosorbent assay (ELISA) plate reader at 570 nm.

Results

The growth-inhibition effects of matrine on U937 cells at various concentrations for 24 h, 48 h and 72 h were determined with the MTT assay. As shown in Fig. [2] A, exposure of U937 cells to matrine at doses less than 0.2 mg/mL for various periods did not cause significant growth inhibition. However, treatment with matrine at 0.4 mg/mL for 24 h resulted in slight growth inhibition and a clear time-intensity of response relationship was observed. In addition, growth inhibition showed a threshold concentration between 0.2 and 0.4 mg/mL in the 72 h treatments.

Based on the observation of proliferation inhibition, induction of apoptosis of U937 cells by matrine was determined by annexin V-FITC/propidium iodine FACS and DNA laddering assays as well as PARP cleavage. As shown in Fig. [2] B, treatment of U937 cells with 0.2 mg/mL of matrine for 48 h and 0.4 mg/mL for 24 h did not cause an increase in the proportion of apoptotic cells, whereas 0.4 mg/mL matrine for 48 h increased the portion of apoptotic cells to 28 %. Similar results were obtained when apoptosis was monitored through the cleavage of PARP (Fig. [2] D). Moreover, exposure of U937 cells to 0.4 mg/mL of matrine for 48 h resulted in DNA laddering indicative of apoptosis (Fig. [2] C).

To determine the mechanism involved in the induction of apoptosis in U937 cells treated with matrine, activation of the caspase cascades (caspase -3, -9, and -7) and release of cytochrome C were investigated. As shown in Fig. [3] A, incubation of the U937 cells for 48 h with 0.4 mg/mL of matrine resulted in the proteolytic cleavage of procaspase-3 to an active cleaved product P19 and P17. The increase in the level of an active P20, which is the cleaved products of procaspase-7, was observed to be proportional to the processed caspase-3. In addition, the cleavage of procaspase-9 into the product P37/35 was induced by matrine concomitantly with the cleavage of procaspase-3. Fig. [3] B shows the release of cytochrome C from the mitochondria into the cytosol after being exposed to matrine. Notably, an increased level of cytochrome C was detected in the cytosol after treatment with 0.4 mg/mL of matrine for 48 h. In parallel, the amount of cytochrome C in the mitochondrial fraction decreased. The observations were determined quantitatively in Fig. [3] C.

The expression of bcl-2 family members is often linked with cytochrome C release in apoptosis induction. As shown in Fig. [3] B and Fig. [3] D, no significant changes in the protein levels of the bcl-2 family members, including bcl-2, bcl-xL and bax, were found in the U937 cells after matrine treatment.

To determine whether the death induction by matrine in U937 cells was associated with activation of MAPKs, we observed the phosphorylation of the three MAPK subfamilies in U937 cells exposed to 0.4 mg/mL of matrine for various times. As shown in Fig. [4], phosphorylation of the three MAPK subfamilies was unchanged in the U937 cells after administration of 0.4 mg/mL of matrine.

Discussion

Our results show that matrine is cytotoxic against U937 cells and induces apoptosis. The results showed that matrine at a dose of 0.4 mg/mL led to toxic effects and it seemed that the dose is too high for anti-cancer drugs. However, in one of the initial studies to treat hepatitis patients in China, matrine was used at a dose of 600 mg daily intravenously for 45 days and subsequently 400 mg daily intramuscularly for 45 days [2]. Compared to other natural products with antileukemia effects, such as homoharringtonine, matrine has few side effects after a long duration of treatment. Moreover, the treatment time of homoharringtonine in clinics is apparently shorter than that of matrine [14]. Here, the treatment of U937 cell with matrine lasted for only 2 or 3 days. Thus it appears worthwhile to study the effects of matrine and its underlying mechanisms at this high dose in vitro.

Matrine inhibits the growth of U937 cells in a time-dependent manner. In addition, treating U937 with matrine for 72 h showed a concentration threshold between 0.2 and 0.4 mg/mL. This effect is different from previous results on the growth inhibition by matrine of K562 cells, which displayed a dose-dependent effect, suggesting that the cell types respond differently to matrine. The phenomenon is consistent with the findings on the effect of several other antileukemia agents such as imatinib on leukemia cells in vitro [15]. In addition, when tested using apoptosis markers such as the proportion of apoptotic cells or the cleavage of PARP, the response of U937 cells to matrine did not show a clear dose-dependency (data not shown).

Caspases play an essential role in apoptosis induced by a variety of stimuli. Caspases are widely distributed in cells in the inactive form and the cleavage of procaspases into active subunits is the response to an apoptosis stimulus. There are two well-established pathways of caspase activation for propagating death signals [16], [17]. One is the activation of caspase-8 mediated by death receptors. Activated caspase-8 processes the downstream effector caspases inducing a cascade of caspase activation. Another pathway involves mitochondria, which release cytochrome C and other proapoptotic proteins from the intermembrane space to the cytoplasm, leading to the activation of caspase-9 and subsequently its effector caspases. Both pathways result in the activation of the major downstream effector capase-3, which cleaves various cellular targets such as poly-(ADP)ribose polymerase and leads to cell death [18]. As shown in Fig. [2] A, the treatment of U937 cells with matrine led to cleavage of caspase-3, -9, -7 into active subunits. In line with these results, PARP, the substrate of caspase-3, was also cleaved into p85 during the process. As these results suggested that matrine might trigger the caspase cascades through the mitochondria in U937 cells [19], [20], the release of cytochrome C from mitochondria to cytosol was measured. As shown in Fig. [2] B and 2C, release of cytochrome C from mitochondria to cytosol occurred in U937 cells exposed to matrine. Taken together, these data suggest that mitochondria as well as downstream caspase cascades were involved in matrine-induced apoptosis.

Intensive studies on apoptosis induction by a variety of stimuli have indicated that bcl-2 family members participate in the regulation of apoptosis [21]. More recently, bcl-2 family proteins including anti-apoptotic bcl-2 and bcl-xL and pro-apoptotic bax have been found to control apoptosis by regulation of cytochrome C release [22], [23], [24], [25]. The participation of bcl-2 family members in apoptosis induction is dependent on both chemotherapeutic agents and cell types. Matrine did not alter the protein levels of bcl-2, bcl-xL and bax. In addition, we did not find a significant translocation of bax from mitochondria to cytosol (data not shown). Whether the controlling of cytochrome C release correlates with other bcl-2 family members in matrine-induced apoptosis in U937 cells remains to be elucidated.

MAPK signaling pathways play critical roles in the regulation of cell survival in U937 cells exposed to various cytotoxic agents. Activities of MAPK are shown in distinct forms involved in apoptosis induction. For instance, JNK is activated in the cell death caused by proteasome inhibitor and CDDO-Me induced phosphorylation of p38 [26], [27]. Both activation of P38 and JNK are required during cantharidin-induced apoptosis in U937 cells [28]. In addition, risedronate suppressed the phosphorylation of Erk 1/2 in apoptosis induction [29]. However, phosphorylations of MAPK were not affected during matrine-induced apoptosis in U937 cells.

In summary, matrine exerts cytotoxicity against U937 cells and stimulates the molecular cascade of apoptosis. The apoptosis induction appears to proceed through MAPK-independent pathways. Further study to explore the exact mechanism involved in the process appears warranted.

Acknowledgements

Thanks are due to Dr. Jun Yin, Shantou University, for the gift of U937 cells. Financial support by the National Natural Science Foundation of China 30 472 185, the Natural Science Foundation of Guangdong Province, P.R.China 04 106 123.

References

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Jikai Jiang

Center for Molecular Biology

School of Medicine

Shantou University

22 Xin Ling Rd

Shantou

Guangdong 515031

People's Republic of China

Phone: +86-754-890-0203

Fax: +86-754-890-0203

Email: jkjiang@stu.edu.cn

References

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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 23 Chipuk J E, Bhat M, Hsing A Y, Ma J, Danielpour D. Bcl-xL blocks transforming growth factor-beta 1-induced apoptosis by inhibiting cytochrome C release and not by directly antagonizing Apaf-1-dependent caspase activation in prostate epithelial cells.  J Biol Chem. 2001;  276 26 614-21
  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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Jikai Jiang

Center for Molecular Biology

School of Medicine

Shantou University

22 Xin Ling Rd

Shantou

Guangdong 515031

People's Republic of China

Phone: +86-754-890-0203

Fax: +86-754-890-0203

Email: jkjiang@stu.edu.cn