Tetramethylpyrazine Attenuates Adriamycin-Induced Apoptotic Injury in Rat Renal Tubular Cells NRK-52E

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Materials
• The cell culture
• Western blot analysis
• Detection of intracellular ROS
• Lactate dehydrogenase assay
• TUNEL staining
• Measurement of the caspase activity
• Determination of TNF-α
• Electrophoretic mobility shift assay (EMSA)
• Statistical analyses
• Results
• Discussion
• References

Abstract

Tetramethylpyrazine (TMP), a compound purified from Rhizoma Ligustici, is a widely used active ingredient in Chinese herbal medicine to treat cardiovascular diseases on account of its vasodilatory actions and antiplatelet activity. Studies have shown that TMP can remove oxygen free radicals and protect rat kidney from ischemia-reperfusion injury. In addition, adriamycin-induced nephrosis in rats is commonly used in pharmacological studies of human chronic renal diseases. Apoptosis of renal tubular cells has been reported in adriamycin-treated rats. To examine the therapeutic potential of TMP on chronic progressive renal diseases, adriamycin-induced injury in rat renal tubular cells NRK-52E has been used to monitor its protective effect. In TUNEL staining, TMP showed a dose-dependent protective effect against adriamycin-induced apoptosis in NRK-52E cells. Pretreatment of the cells with 10 or 100 µM of TMP effectively decreased the reactive oxygen species (ROS) formation induced by adriamycin, as measured in fluorescent assays. TMP was found to reduce the adriamycin-stimulated activities of caspase-3, caspase-8 and caspase-9, inhibit adriamycin-induced release of cytochrome c, and elevate the expression of Bcl-x_L. TMP was also able to inhibit the death receptor signaling pathway and suppress the activation of transcription factor NF-κB in adriamycin-treated NRK-52E cells. Based on the results of this study, we suggest that TMP can attenuate adriamycin-induced oxidative stress and apoptotic injury in NRK-52E cells, and that it may have therapeutic potential for patients with renal diseases.

Abbreviations

TMP: tetramethylpyrazine

LDH: lactate dehydrogenase

ROS: reactive oxygen species

DCF: 2′,7′-dichlorofluorescein

TNF-α: tumor necrosis factor-α

TUNEL: terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling

Introduction

Tetramethylpyrazine (TMP, also named Chuan Xiong ), a compound purified from the rhizome of Ligusticum wallichi, is a widely used active ingredient in Chinese herbal medicine to treat cardiovascular diseases and stroke [1], [2], [3]. Several studies have shown that TMP can remove oxygen free radicals, probably due to increased activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) [4], [5], [6], [7]. TMP was also found to protect rat kidneys from ischemia-reperfusion injury [8], [9]. Those investigations suggest that TMP reduces the renal dysfunction associated with warm I/R of the kidney. Additionally, adriamycin is the anti-tumor anthracycline antibiotic of choice for the treatment of many solid malignancies and lymphomas. Adriamycin-induced nephrosis in rats is used to study human chronic renal diseases [10], [11]. Induction of apoptosis is an important cytotoxic mechanism of adriamycin [12] and apoptosis of renal tubular cells has been reported in adriamycin-treated rats [13]. Renal tubular cell apoptosis with a key feature of tubular atrophy, is a hallmark of chronic renal diseases [14], [15].

Reactive oxygen species (ROS) derived from redox activation of adriamycin have been proposed to be responsible for adriamycin-induced cytotoxicity [16]. The results of previous studies have indicated that, at submicromolar concentrations, adriamycin induces apoptosis with the activation of caspases in endothelial cells and myocytes [17], [18]. In mammalian cells, a major caspase activation pathway is the cytochrome c-initiated pathway. In this pathway, various apoptotic stimuli cause cytochrome c release from mitochondria, which in turn induce a series of biochemical reactions, resulting in activation of caspase to cause subsequent cell death [19]. Cytochrome c release is known to be regulated by Bcl-2 family proteins, including Bcl-2 and Bcl-xL, which bind to the mitochondrial outer membrane and block cytochrome c efflux [20]. In addition to mitochondria-mediated apoptosis signaling, adriamycin also induces the activity of nuclear factor kB (NF-kB) [21], which can regulate the expression of many genes in apoptosis. Therefore, mitochondria-mediated signaling and NF-kB activation play major roles in adriamycin-induced cytotoxicity.

In this study, we intended to evaluate the cytoprotective effect of TMP on adriamycin-induced injury in rat renal tubular cell NRK-52E, and to investigate the anti-oxidative and anti-apoptotic effects of TMP in adriamycin-treated NRK-52E cells.

Materials and Methods

Materials

Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, and tissue culture reagents were from Life Technologies, Inc (Gaithersburg, MD, USA). Tetramethylpyrazine was purchased from Aldrich (St. Louis, MO, USA). All other chemicals of reagent grade were obtained from Sigma (St. Louis, MO, USA). Anti-mouse Fas activating antibody was purchased from MBL Co. (Nagoya, Japan).

The cell culture

Rat renal proximal tubular cells (NRK-52E) were purchased from Bioresource Collection and Research Center (BCRC; Hsinchu, Taiwan), and cultured in DMEM culture medium supplemented with antibiotic/antifungal solution and 10 % fetal bovine serum. They were grown until the monolayer became confluent. The medium for the cultured cells was then changed to the serum-free medium, and the cells were incubated overnight before the experiment. MCF-7 human breast cancer cells from BCRC were cultured in DMEM containing 4.5 g/L glucose and supplemented with 10 % fetal calf serum and antibiotics. Cells were subcultured twice weekly and 48 h before the initiation of an experiment.

Western blot analysis

We applied 30 μg of NRK-52E lysate proteins to each lane and analyzed them with Western blots. Cytosol cytochrome c was extracted by using the Cytochrome c Release Apoptosis Assay Kit (Calbiochem Inc.; Darmstadt, Germany). The antibodies of caspase-3, caspase-8, caspase-9, cytochrome c, Bcl-x_L and NF-κB were purchased from BD Laboratories (San Jose, CA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), and diluted to 1 : 1000 for assay. We used peroxidase-conjugated anti-rabbit or anti-mouse IgG (1 : 5000 dilution) as the secondary antibody to detect caspase-3, caspase-9, cytochrome c, Bcl-2 and NF-κB bands by enhanced chemiluminescence (Amersham; Pittsburgh, PA, USA).

Detection of intracellular ROS

Prior to the chemical treatment, NRK-52E were incubated in culture medium containing a fluorescent dye, 2′,7′-dichlorofluorescein (DCF) of 30 μM for 30 minutes to establish a stable intracellular level of the probe. The same concentration of DCF was maintained during the chemical treatment. Subsequently, the cells were washed with PBS, removed from Petri dishes by scraping, and measured for DCF fluorescence intensity. We determined the DCF fluorescence intensity of the cells by fluorescence spectrophotometry with excitation and emission wavelengths at 475 and 525 nm, respectively. The cell number in each sample was counted and used to normalize the fluorescence intensity of DCF. We also performed a chemiluminescence assay of superoxide production. NRK-52E cells were lysed after drug treatment with a lysis buffer containing lucigenin (200 μM). Readings were started immediately upon addition of the lysis buffer. We used samples with the addition of SOD (1.0 × 10⁵ U/L) as blank controls. Each reading was recorded as single photon counts by using a microplate scintillation counter (Topcount, Packard Instrument Co.; Meriden, CT, USA).

Lactate dehydrogenase assay

For lactate dehydrogenase (LDH) assays, NRK-52E cells were plated at 10,000 cells/well in 96-well plates and grown overnight. After treatment with adriamycin, we collected and assayed the culture medium with the LDH Cytotoxicology Detection kit (Roche; Mannheim, Germany) according to the manufacturer's directions. Each data point was determined in triplicate.

TUNEL staining

Adriamycin-mediated apoptosis in NRK-52E cells was detected by enzymatic labeling of DNA strand breaks using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining. TUNEL staining was conducted using a Cell Death Detection kit (Roche; Mannheim, Germany) and performed according to the previous study [22].

Measurement of the caspase activity

We determined caspase-3 activity spectrofluorometrically using the synthetic substrate Ac-DEVD-AFC (BIOMOL Research Labs; Dural, Australia). Cell homogenates were incubated with synthetic substrate at 0.2 mM at 37 °C/2 h. The fluorescence intensity of liberated AFC was monitored using a fluorescence spectrophotometer at an excitation wavelength of 400 nm and an emission wavelength of 505 nm.

Determination of TNF-α

NRK-52E cells were cultured on 10-cm plates with 2 mL medium in each plate, pretreated with TMP for 30 min or without, and then treated with adriamycin at 3 μM for 8 h. The culture medium was collected and analyzed using the mouse TNF-α ELISA kit according to instructions provided by the manufacturers (RayBiotech, Inc.; Norcross, GA, USA).

Electrophoretic mobility shift assay (EMSA)

To prepare nuclear protein extracts, we washed cultured NRK-52E with cold PBS and then immediately removed them by scraping in PBS. After centrifugation of the cell suspension at 2000 rpm, the cell pellets were resuspended in cold buffer A (containing KCl 10 mM, EDTA 0.1 mM, DTT 1 mM and PMSF 1 mM) for 15 min. We lysed the cells by adding 10 % NP-40 and then centrifuged them at 6000 rpm to obtain pellets of nuclei. The nuclear pellets were resuspended in cold buffer B (containing HEPES 20 mM, EDTA 1 mM, DTT 1 mM and PMSF 1 mM and NaCl 0.4 mM), vigorously agitated, and then centrifuged. The supernatant containing the nuclear proteins was used for Western blot analysis or stored at -70 °C until use. A double-stranded probe containing a high affinity sequence for NF-κB from the mouse kappa-light chain enhancer (5′-AGC TTC AGA GAC TTT CCG AGA GG-3′) was prepared. The oligonucleotide was end labeled with [³²P]ATP. Extracted nuclear proteins (10 μg) were incubated with 0.1 ng ³²P-labeled DNA for 15 min at room temperature in 25 μL binding buffer containing 1 μg poly (dI-dC). We electrophoresed the mixtures on 5 % non-denaturing polyacrylamide gels. Gels were dried and imaged by autoradiography.

Statistical analyses

Data were presented as mean ± standard deviation (S.D.), and groups were compared using the analysis of variance (ANOVA). The differences were considered significant if the p value was smaller or equal to 0.05.

Results

To determine the cytotoxicity of adriamycin on rat renal tubular cells NRK-52E, the lactate dehydrogenase (LDH) released from the cytosol of damaged cells was measured. NRK-52E cells were treated with 3μM of adriamycin from 6 to 24 hours. As shown in Fig. [1] A, exposure to adriamycin for 6 hours markedly increased LDH leakage into medium, and the cytotoxicity of adriamycin increased during the administration time. Similar results were also found in TUNEL staining (Fig. [1] B). These data reveal that adriamycin-induced injury in NRK-52E cells is highly related to apoptosis. Subsequently, the protective effect of tetramethylpyrazine against the apoptotic cytotoxicity was examined by using TUNEL staining. As shown in Fig. [1] C, tetramethylpyrazine reduced adriamycin-induced apoptosis in NRK-52E cells in a concentration-dependent manner. It should be noted that adriamycin represents an established drug for the treatment of tumors. The influence of tetramethylpyrazine on adriamycin cytotoxicity in cancer cells was also determined using LDH assays with MCF-7 human breast cancer cells. The result showed that tetramethylpyrazine barely influenced adriamycin-induced cell death in MCF-7 cells; even 100 μM of tetramethylpyrazine only slightly reduced adriamycin-induced cytotoxicity (Fig. [1] D). These data imply a therapeutic potential for tetramethylpyrazine.

We next examined whether tetramethylpyrazine prevents adriamycin-induced ROS formation. NRK-52E cells were treated with tetramethylpyrazine (1 - 100 μM) in the absence or presence of adriamycin. Adriamycin-induced increases in intracellular ROS were revealed by fluorescent intensities of 2′,7′-dichlorofluorescin (DCF). Tetramethylpyrazine significantly inhibited adriamycin-induced ROS formation under treatment of 3 μM adriamycin for 24 h (Fig. [2] A). A chemiluminescence assay of superoxide production also showed a similar result (Fig. [2] B).The effect of tetramethylpyrazine on adriamycin-induced activation of caspases was evaluated by Western blotting using antibodies that recognize either full-length (procaspase-8 and procaspase-9) or cleaved (caspase-3) caspases. The 19 kDa intermediate of caspase-3 was greatly elevated in the cells treated with 3μM of adriamycin for 24 hours, and the prototypes of caspase-8 and caspase-9 were reduced. Pretreatment with tetramethylpyrazine at 10 or 100 μM for 30 minutes significantly reduced the quantity of caspase-3 and increased the quantity of procaspase-8 and procaspase-9, in comparison with that in cells treated with adriamycin alone (Fig. [3] A). In the caspase-3 activity assay, the adriamycin-induced activity of caspase-3 was also reduced by tetramethylpyrazine treatment (Fig. [3] B).

The reduction of procaspase-9 is associated with mitochondria-mediated signaling pathway in apoptosis. To evaluate the influence of tetramethylpyrazine on the variations of mitochondria-mediated signaling molecules caused by adriamycin, such as cytochrome c and Bcl-x_L, NRK-52E cells pretreated with tetramethylpyrazine were treated with 3μM adriamycin for 6 hours. Western blotting was carried out with the specific antibody against cytochrome c and Bcl-x_L. In the adriamycin alone treatment group, cytosol cytochrome c was increased significantly, and the expression of Bcl-x_L was reduced (Fig. [4]). However, the variations of cytosol cytochrome c and Bcl-x_L were inhibited by tetramethylpyrazine in a dose-dependent manner.

Additionally, the reduction of procaspase-8 is associated with death receptor-mediated apoptosis. To confirm the inhibitory effect of tetramethylpyrazine on death receptor-mediated signaling, anti-Fas activating antibody was applied to induce death receptor-mediated apoptosis in NRK-52E cells. As shown in Fig. [5] A, tetramethylpyrazine inhibited the reduction of procaspase-8 caused by anti-Fas antibody. Tumor necrosis factor-α (TNF-α) is an important mediator for death receptor-mediated apoptosis, and its excretion caused by adriamycin was also inhibited by tetramethylpyrazine (Fig. [5] B). We further monitored the activation of the transcription factor of TNF-α, NF-κB, in adriamycin-treated NRK-52E cells. The DNA-binding activity of nuclear NF-κB was monitored by using the electrophoretic mobility shift assay (EMSA) and it is responsible for the majority of the NF-kB activity. As shown in Fig. [6], the increase of DNA binding activity of NF-κB by adriamycin was reduced significantly by tetramethylpyrazine in a dose-dependent manner.

Discussion

The major findings in our study showed that tetramethylpyrazine attenuated adriamycin-induced apoptotic injury in rat renal tubular cell NRK-52E (Fig. [1]), that ROS formation induced by adriamycin caused adriamycin-induced cytotoxicity [16] and that the free radicals might lead to apoptosis. As shown in Fig. [2], tetramethylpyrazine was found to reduce the ROS formation in adriamycin-treated NRK-52E cells, which may be an important mechanism of tetramethylpyrazine's protective function. However, the influence of tetramethylpyrazine on adriamycin-induced cytotoxicity was limited in MCF-7 cancer cells (Fig. [1] D). This implies the therapeutic potential of tetramethylpyrazine in cancer therapy although the mechanism of the different sensitivity between normal cells and cancer cells is not clear yet. We also monitored the apoptotic signals associated with adriamycin in NRK-52E cells, such as the activation of caspases, the increased release of cytochrome c, the Bcl-x_L reduction, and the induction of TNF-α excretion. The results of our study also indicated that those apoptotic signals exist in adriamycin-treated NRK-52E cells, that they are reversed by tetramethylpyrazine treatment. Taken together, we suggest that tetramethylpyrazine achieves its protective effects on adriamycin-treated rat renal tubular cells through the inhibition of apoptotic signaling pathways.

Caspase-dependent apoptotic signaling plays a major role in adriamycin-induced apoptotic injury. Caspase-3 is an executioner caspase that can be activated by caspase-9 in the mitochondrial pathway, or caspase-8 in the death receptor pathway [23], [24]. Caspase-9 is activated from procaspase-9 by cytosolic cytochrome c [19]. The mitochondrial release of cytochrome c is regulated by Bcl-2 family proteins, including Bcl-2 and Bcl-x_L, which bind to the mitochondrial outer membrane and block cytochrome c efflux [20]. In this study, adriamycin markedly reduced the Bcl-x_L expression, which was reversed by tetramethylpyrazine treatment. On the other hand, caspase-8 is activated from procaspase-8 by Fas-associated death domain (FADD) protein in the death receptor pathway [25]. It is noteworthy that tetramethylpyrazine inhibited the cleavage of caspase-8 induced by both adriamycin and anti-Fas antibody. Tetramethylpyrazine also inhibited adriamycin-induced TNF-α excretion. Based on the findings of this study, we suggest that tetramethylpyrazine protects renal tubular cells from adriamycin-induced apoptotic injury through inhibition in both the mitochondrial pathway and the death receptor pathway.

NF-κB activation and translocation are pro-apoptotic in adriamycin-treated endothelial cells and cardiomyocytes [21]. NF-κB has also been reported to be involved in regulating adriamycin-induced apoptosis in various cancer cells and carcinomas [26], [27]. As shown in Fig. [6], the findings of our study showed that TMP inhibited adriamycin-induced NF-κB activation in rat renal tubular cells. The pro-apoptotic character of NF-κB might be due to its direct activation of apoptotic genes, including TNF-α, Fas ligand, c-Myc and p53 [28], [29]. In addition, the findings in recent studies reveal that H₂O₂ is responsible for adriamycin-induced NF-kB activation in adriamycin-treated endothelial cells and cardiomyocytes [21]. Tetramethylpyrazine can inhibit ROS production, resulting in inhibition of NF-κB activation induced by adriamycin as shown in our results.

In summary, tetramethylpyrazine can reduce the adriamycin-induced ROS formation and NF-κB activation as well as inhibit both mitochondria-mediated and death receptor-mediated apoptotic pathways. Through these mechanisms, tetramethylpyrazine may protect rat renal tubular cells from adriamycin-induced apoptosis.

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Yung-Ho Hsu, MD

Department of Medicine

Taipei Medical University-Wan Fang Hospital

No 111 Sing-Lung Road Sec. 3

Wen-Shan District

Taipei 116

Taiwan

Republic of China

Phone: +886-2-2930-7930 ext. 2711

Fax: +886-2-2933-4920

Email: yhhsu@tmu.edu.tw

References

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  PubMedSearch in Google Scholar
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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
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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Yung-Ho Hsu, MD

Department of Medicine

Taipei Medical University-Wan Fang Hospital

No 111 Sing-Lung Road Sec. 3

Wen-Shan District

Taipei 116

Taiwan

Republic of China

Phone: +886-2-2930-7930 ext. 2711

Fax: +886-2-2933-4920

Email: yhhsu@tmu.edu.tw