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Involvement of NF-[kappa]B and c-myc signaling pathways in the apoptosis of HL-60 cells induced by alkaloids of Tripterygium hypoglaucum (levl.) Hutch.

Summary

Tripterygium hypoglaucum (levl.) Hutch (Celastraceae) (THH) root is a Chinese medicinal herb commonly used for treating autoimmune diseases. In the present study, alkaloids of THH were prepared and their cytotoxicity against the HL-60 cell was investigated. THH-induced apoptosis was observed using flow cytometry, confocal fluorescence microscope, and DNA laddering and caspase assays. The molecular mechanism involved in the induction of HL-60 cell apoptosis by THH alkaloids was examined using cDNA microarrays containing 3000 human genes derived from a leukocyte cDNA library. Sixteen genes were identified to be differentially expressed in HL-60 cells upon THH treatment. Several genes related to the NF-[kappa]B signaling pathway and cell apoptosis (such as NFKBIB, PRG1 and B2M) were up-regulated. In addition, c-myc binding protein and apoptosis-related cysteine proteases caspase-3 and caspase-8 were also regulated. The changes in c-Myc RNA expression and c-myc protein level were further confirmed by RT-PCR and Western blot analysis. The results demonstrated that THH alkaloids induced apoptosis of HL-60 cells though c-myc and NF-[kappa]B signaling pathways.

Key words: Tripterygium hypoglaucum (levl.) Hutch extracts, cytotoxicity, apoptosis, cDNA microarray

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Introduction

The root of Tripterygium hypoglaucum (levl.) Hutch (THH) has been used in traditional Chinese medicine to treat auto-immune diseases, including rheumatoid arthitis, systemic lupus erythomatosis and skin problems. Preliminary studies have shown that THH affected T lymphocytes (Zhao, 1992) and induced chomosome changes in mouse bone marrow cells (Wang et al. 1993). It was reported that THH caused apoptosis in a number of cultured cells, for example, acute T cell leukemia cells, Jurkat, CHE and NIH3T3 cells, with leukemia cells being the most sensitive (Cao and Nusse, 1999). Apoptosis is a form of physiological cell death essential to normal tissue development and homeostasis (Kerr et al. 1972). After receiving an apoptotic death stimulus, cells first enter a signaling phase followed by the final degradation phase, in which apoptosis is identifiable by chomatin condensation, cell shinkage, caspases activation, membrane lipid rearrangement, DNA fragmentation ("DNA-laddering") and cell fragmentation, though the formation of "apoptotic bodies" (Jacobson et al. 1997).

HL-60 is a human promyelocytic leukemia cell line that originated from a patient with symptoms of acute myeloid leukemia (Collins et al. 1977) and can be induced to differentiate and/or to enter apoptosis by a variety of pharmacological agents, including Retinoic acid and Hexamethylenebisacetamide (HMBA) (Hozumi, 1998). In this study, the cytotoxic effect of THH alkaloids on the HL-60 cell line was examined, and the mechanism of action was investigated using a number of molecular and cellular techniques.

Material and Methods

Preparation of THH alkaloids

Dried root of T. hypoglaucum (levl.) Hutch (THH) is readily available from most well stocked herbal outlets in China. A single lot of dry THH root (Yunnan Medicine Company, Kunming, China) was used in this study. Dry root powder was soaked in 60% ethanol for 6 days, and the extract was filtered, concentrated, and acidified with HCl. The filtrate was further extracted with ether and chloroform, and the organic layer was collected and dried. The dried products were dissolved in DMSO and irradiated by Co-60, and stored at -20[degrees]C for long-term storage.

Characterization of THH alkaloids by HPLC

THH alkaloids were dissolved in methanol and subjected to HPLC analysis. The result is shown in Figure 1.

Cell culture and treatment of THH alkaloids

HL-60 cells (American Type Culture Collection, MD, USA) were routinely grown at 37[degrees]C in 5% C[O.sub.2] air in RPMI-1640, supplemented with 1% antibiotic solution and 10% heat-inactivated fetal bovine serum (Invitrogen, Gaithersburg, MD).

[FIGURE 1 OMITTED]

For THH alkaloid treatment, HL-60 cells were allowed to grow for 72 h after sub-culture, and growth medium was then replaced with a half volume of fresh medium, with different concentrations of THH extraction for dose-course assay or addition of THH to a final concentration of 40 [micro]g/ml, in culture medium, for time-course assay.

Flow cytometry and confocal microscopy

Approximately 2-3 X [10.sup.6] of control or THH-treated cells were collected by centrifugation and washed twice with phosphate-buffered saline (PBS). The cell pellets were resuspended gently in 1 ml of hypotonic propidium iodide (Sigma chemicals, St. Louis, MO) solution (50 [micro]g/ml) prepared in 0.1% sodium citrate plus 0.1% Triton X-100 and 100 mg/ml DNase-free RNase A. The stained cells were analyzed using a flow cytometry (ESP, Coulter Electronic, USA) at excitation wavelength of 488 nm and emission wavelength of 600 nm. Data were analyzed by cycle distribution software (ModFit LT version 2.0, Verity Software House, USA).

Cells were fluorescence labeled by adding 40 [micro]l staining solution (100 [micro]g/ml each of acridine orange and ethidium bromide) to 1ml cell suspension. After 3-5 min, cells were studied using a confocal laser-scanning microscope (LSM510, Carl Zeiss) at an excitation wavelength of 490 nm and an emission wavelength of 510 nm.

DNA ladder analysis

Approximately 2.5 X [10.sup.6] cells were washed in PBS and incubated in lysis-buffer (10 ml Tris, pH 7.5-8.0, 1 mM EDTA, 0.5% Triton X-100) at 4 [degrees]C for 30 min. Cell lysates were centrifuged (25 000 X g, 20 min) and the supernatant was treated with RNase A (300 [micro]g/ml, 37 [degrees]C, 60 min) and proteinase K (200 [micro]g/ml, 37[degrees]C for 60 min). After phenol/chloroform extraction and centrifugation (20 000 X g, 5 min), DNA was precipitated by adding 95% ethanol and stored at -20 [degrees]C overnight. DNA was collected by centrifugation (4 [degrees]C, 29 000 X g, 20 min) and analyzed by electrophoresis in 1.2% agarose gel containing 0.1% ethidium bromide in TAE buffer (60 v, 2 h). DNA bands were observed and recorded using a Multi-Imager (Bio-Rad, California, USA).

Caspase activity assay

Caspase-8 and caspase-3 were monitored using fluorometric assay kits (Clontech, California, USA). Approximately 2.5 X [10.sup.6] treated cells were treated with the lysis buffer provided with the kits, and then IETD-pNA (caspase-8 substrate) or DEVD-AFC (caspase-3 substrate) was added and incubated at 37 [degrees]C for 1 h. Samples were placed in 96-well plates and read by fluorometer (excitation 360 nm/emission 450 nm) (F/Max Molecular Devices, US).

cDNA microarray hybridization

A total of 3000 genes and ESTs from a human leukocyte cDNA library (Clontech, California, USA) was amplified with the polymerase chain reaction (PCR) and arrayed on the glass slides using a microarray printer SPBIO (Hitachi, Japan). The arrays were processed by chemical and heat treatment to attach the DNA fragments to the glass surface, according to published protocols (Hegde et al. 2000).

HL-60 cells untreated (control) and treated with alkaloids of THH (40 [micro]g/ml, 8 h) were harvested and collected in a 50-ml conical tube by centrifugation (1000 rpm, 5 min). Cells were lysated with TRIZOL reagent (Invitrogen, Gaithersburg, MD) and total RNA was isolated according to the manufacturer's protocol. The concentration of total RNA was measured with a biophotometer (Eppendorf, Germany) and the RNA quality was verified using a 1% denatured agarose gel. The same amounts of total RNA from control or treated total RNA were reverse transcripted to cDNA. During the reaction, two distinct fluorescent dyes, Cy3 and Cy5, were labeled with control and treated samples, respectively. The labeled cDNA was then purified using Microcon 30 (Millipore, Japan) and mixed with hybridization buffer before application to a prepared microarray. Hybridization was carried out at 65 [degrees]C overnight. Microarray images were obtained using a confocal fluorescence scanner (ScanArray 4000, GSI Lumonics, USA) and the scan results were analyzed using ScanAnalyze software (Standford University, California, USA). Fluorescence ratios (Cy5 vs. Cy3) were used to determine the differential gene expression levels.

Northern blot hybridization

10 [micro]g of total RNA isolated from THH-treated (40 [micro]g/ml, 8h) and untreated HL-60 cells were processed on formaldehyde denaturing 1% agarose gel. The gels were blotted onto nylon membrane (Hybond-[N.sup.+], Amersham). The membrane was UV-cross-linked and hybridized with [.sup.32]P-labeled probes, e.g. proteoglycan 1, secretory granule (PRG1) and Cyclin B2 (CCNB2). The relative amount of mRNA was determined by autoradiography. A housekeeping gene (ACTIN) was used as a control.

RT-PCR (reverse transcription-polymerase chain reaction) of c-myc

Total RNA (1 [micro]g) isolated from cultured cells using High Pure[TM] RNA Isolation Kits (Roche, Germany) was incubated with 100ng random primers (Gibco BRL, MD, USA) and RNase-free water at 65 [degrees]C for 10 min, then placed on ice for 5 min. The reverse transcription mixture contained 4 [micro]l of 5X reaction buffer (50 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 10 mM DTE, 0.05% polydocanol v/v, 50% glycerol v/v, pH 8.4), 2 [micro]l 10 mM dNTP, 2 [micro]l 100 mM ditheitol and 1 ml 50 units/ml Expand[TM] Reverse Transcriptase (Roche, Germany). The reaction lasted 1.5 h at 42 [degrees]C. PCR amplifications were carried out using Expand[TM] Long Template PCR System (Roche). Primers were human c-myc cDNA sequences 5'-CCTACCCTCTCA ACGACAGC-3' (sense) and 5'-GTTGTGTGTTCGC CTCTTGA-3' (antisense); human [beta]-actin cDNA sequences 5'-GATGATATCGCCGCGCTCGTCGTCG AC-3' (sense) and 5'-AGCCAGGTCCAGACGCAG GATGGCATG-3' (antisense). Five [micro]Ci of [[alpha]-[.sup.32]P]dCTP were added to 25 [micro]l amplification reaction mix. PCR was performed for 22 cycles, consisting of 1 min at 94 [degrees]C, 1 min at 60 [degrees]C and 1 min at 72 [degrees]C. The samples were heated for 5 min at 94 [degrees]C before the first cycle, and the extension time was lengthened to 10 min during the last cycle. PCR products were size-fractionated on 10% polyacrylamide gel. Autoradiography was carried out with BioMax film (Kodak, USA).

Western blot analysis of c-myc expression

Whole cell extracts containing 15 to 20 [micro]g protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane. After blocking for 1 h with Tris-buffer saline containing 5% nonfat milk powder, filters were incubated with mouse anti human c-Myc monoclonal antibody Ab-3 (1:80 dilution) (Calbiochem, California, USA). Filters were washed for 1 min with Tri-buffered saline containing 5% BSA and 0.1% Tween 20, and incubated for 1 h with horseradish peroxidase-conjugated goat antimouse immunoglobulin G secondary antibodies (Bio-Rad, California, USA). Blots were washed 6 X 5 min with Tris-buffered saline containing 5% nonfat milk powders and 0.1% Tween 20. Tagged protein bands were detected by treatment with ECL Western blotting reagents (Amersham, NJ, USA) and exposure on X-ray films (Fuji, Japan).

Results and Discussion

THH alkaloids induced apoptosis in HL-60 cells

Flow cytometry was used to study the effect of THH alkaloids on HL-60 cells. Cells were treated with 0 (control), 25 [micro]g/ml and 40 [micro]g/ml of THH alkaloids for 8 h. As shown in Fig. 2, a distinct sub-G1 hypodiploid peak was observed for cells treated with 40 [micro]g/ml THH alkaloids (Fig. 2c), as compared to control (Fig. 2a) and 25 [micro]g/ml THH treated cell (Fig. 2b). The accumulation of dying cells in this "sub-G1" hypodiploid peak is mainly due to reduced DNA content, and these cells are destined to enter apoptosis. After cell cycle distribution analysis, we also found that the percentage of [G.sub.0]/[G.sub.1] phase increased with the increasing of THH alkaloids concentration; by contrast, the S phase reduced significantly (Table 1).

[FIGURE 2 OMITTED]

The morphology of cells was examined using a fluorescence confocal laser scanning microscope, where cells were stained with acridine orange/ethidium bromide (Fig. 3A). Untreated normal HL-60 cells (Fig. 3A-a) appeared uniformly green with distinct round nucleoli. After treatment with 25 [micro]g/ml THH for 8 h, cell shinkage, chomatin condensation and nuclear fragmentation became obvious in some of the cells (Fig. 3A-b). After treatment with 40 [micro]g/ml THH for 8 h, cellular fragmentation resulted in the appearance of apoptotic bodies (Fig. 3A-c).

DNA laddering analysis was also used to further elucidate the apoptotic effect of THH on HL-60 cells. As shown in Fig. 3B, oligonucleosome DNA fragments with sizes in multiples of ~ 180 base pair (DNA laddering) were detected in cells after 8 h treatment by 40 [micro]g/ml THH alkaloids.

Activation of caspases, a family of cysteine proteases which specifically cleave at aspartic acid residues, is central to the execution of apoptosis (Thornberry and Lazebnik, 1998; Nagane et al. 2001). In order to further investigate the change of caspase activity during THH treatment, caspase activity assays were used to study caspase-3 and caspase-8 activities involved in the route of activation of TRAIL. Caspase-3 activity increased in THH-treated HL-60 cells (40 [micro]g/ml) and peaked at about 12 h (Fig. 4A). On the other hand, caspase-8 activity in HL-60 cells increased and reached the maximum level after 8 h of THH treatment (40 [micro]g/ml, Fig. 4B). This is expected, because caspase-8 is a major initiation in death receptor signaling; active caspase-8 leads to the activation of downstream caspases, including caspase 3 (Chen and Wang, 2002). Our result confirmed that the HL-60 cells entered into apoptosis after THH treatment. The process was probably executed by a caspase-8 activated proteolytic cascade and caspase-3 plays a direct role in irreversible cellular destruction.

The above results provide c lear evidence that THH alkaloids caused apoptosis in HL-60 cells. Characteristic apoptotic features were induced by THH alkaloids after a short exposure time, suggesting a specific pharmacological action of THH.

Gene expression profiles of HL-60 cells upon THH treatment

The cDNA microarray technique has become a valuable and powerful tool for generating information on the expression profiles of thousands of genes simultaneously (Kurian et al. 1999). In order to investigate the molecular mechanisms responsible for THH-induced apoptosis in HL-60 cells, cDNA microarrays were used in this study to compare the gene expression patterns between untreated cells and cells treated with THH alkaloids. Total RNA from untreated and THH-treated HL-60 cells (40 [micro]g/ml, 8 h) was used as templates for synthesis of Cy3 (green) and Cy5 (red) labeled cDNA probes, respectively. Figure 5A showed a typical microarray image following the competitive hybridization of the two differently labeled cDNA with the cDNA microarray containing 3000 genes and ESTs amplified from a human leukocyte cDNA library. Image analysis showed that 16 genes displayed a greater than 2-fold difference between untreated and THH-treated samples. Table 2 lists the accession numbers, functional description, as well as the Cy5/Cy3 fluorescence ratios of these genes. The differentially expressed genes can be classified into thee main categories: apoptosis, cell cycle and differentiation, and stress response (Table 2). Both caspase-3 and caspase-8, important common genes in executing the apoptosis process, were up-regulated, which is consistent with the results obtained from caspase activity measurements.

[FIGURE 3 OMITTED]

Northern blot hybridization experiments were carried out to verify the microarray results, using the same total RNA samples prepared for microarray studies. Probes for proteoglycan 1, secretory granule (PRG1) and Cyclin B2 (CCNB2), were selected for amplification and radioactive labeling, as they were differentially regulated based on the microarray results. ACTIN, a housekeeping gene with expression unchanged during THH treatment, was chosen to serve as a control. The Northern blot results are shown in Fig. 4B, together with the microarray spots corresponding to the respective genes. The results demonstrated clearly that similar expression levels for the selected genes were obtained by both cDNA microarray and Northern blot hybridization experiments.

[FIGURE 4 OMITTED]

THH-induced apoptosis involved NF-[kappa]B and mitochondrial pathways

Among the apoptosis-related genes, most are involved in two signaling pathways leading to apoptosis, the NF-[kappa]B signaling pathway or the mitochondrial mediated signaling pathway. For example, NFKBIB, the gene encoding the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor beta, was up-regulated by more than four fold. NFKBIB is the central gene in the NF-[kappa]B signaling regulation (Schafer et al. 1996; Athanasios, 1997). Proteoglycan 1, secretory granule (PRG1), an early response gene regulated by P53 and NF-[kappa]B (Schafer et al. 1996), was up-regulated by more than thee fold. Beta-2 microglobulin ([B.sub.2]M), the NF-[kappa]B target gene for immunoreceptor (Schafer et al. 1996), was also up-regulated during THH treatment. On the other hand, ATP synthase 3, a member of the solute carrier (mitochondrial carrier; phosphate carrier) family, and mitochondrial ribosomal protein S12, were both up-regulated, indicating the involvement of the mitochondrial mediated apoptotic pathway. It is well known that apoptosis is an active, ATP-requiring process (Ross et al. 1999).

Two genes were down-regulated, including an NDP kinase protein expressed in non-metastatic cells 5, and c-myc binding protein. Both are involved in the proliferation of cells and it is expected that their activities must be attenuated during apoptosis (Ross et al. 1999). Several cell cycle related genes, such as cyclin B2 and interleukin 10 receptor were down-regulated after THH treatment, consistent with the fact that the HL-60 cells were induced into G1-G2 arrest and apoptosis. Two genes related to cell differentiation, ribosomal protein L31 and a histone family gene were up-regulated, although there is no evidence showing that THH can induce cell differentiation. Several stress response genes, including heat shock 70 kD protein 4 and 90 kD protein 1, beta, were up-regulated during THH treatment, which is a common response mechanism for cells under drug treatment.

Down-regulation of c-Myc

A dual signal model for c-Myc function in growth and apoptosis regulation has been proposed (Rendergast, 1999) that c-Myc activates proliferation and primes apoptosis though only regulation of c-myc expression alone, or in conjugation with a second critical gene (Skew et al. 1991). The consequences of down-regulating c-Myc were studied in myc-null cells (Mateyak et al. 1997), where the doubling time of these cells was prolonged with the accumulations of cells in G1 and G2 phases but not S phase. Treatments that cause apoptotic death of several cell types have been shown to first drastically lower c-myc expression (Sonenshein, 1997; Packham and Cleveland, 1995). Although the down-regulation of c-myc binding protein A was observed from the microarray experiments, c-myc was not included on the microarray. In order to obtain direct evidence on how c-myc is regulated during THH treatment, RT-PCR and Western blot analysis were used to measure the RNA and protein levels of c-myc in HL-60 cells. RT-PCR results showed that the relative levels of c-myc mRNA were 92%, 55% and 9% of the control after 4, 8 and 12 h of THH treatment (40 [micro]g/ml), respectively, after normalization with [beta]-actin mRNA (Fig. 6A). Western blot hybridization revealed that c-myc protein level in the control cell remained high, but the protein level decreased drastically to a level almost undetectable after 2 to 4 h of THH treatment (Fig. 6B). The results indicated that THH alkaloids caused an initial decrease in c-myc protein level in HL-60 cells by translational and/or post-translational regulation, and a subsequent down-regulation of c-myc RNA expression further reduced the c-myc protein level. The direct involvement of c-myc and c-myc binding protein in THH-induced apoptosis also illustrated the importance of c-myc in regulating cell cycle control, proliferation and apoptosis in HL-60 cells (Dang, 1999). The upstream events leading to the changes in c-myc protein and RNA levels upon THH treatment are under investigation.

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

In conclusion, the present study has demonstrated that THH alkaloids induced apoptosis in human promyleocytic leukemia HL-60 cells. The gene expression profiles of HL-60 cells treated with THH (40 [micro]g/ml, 8 h) were generated using cDNA microarray technology. Sixteen genes were identified to be differentially expressed in HL-60 cells upon THH treatment and were clustered in different functional categories, including apoptosis and cell cycle and stress response. Several genes related to NF-[kappa]B and c-myc signaling pathways were regulated by THH. RT-PCR showed a late decrease in c-myc mRNA. On the other hand. Western blot analysis showed an early >99% decrease of the nuclear oncoprotein c-myc after 2 to 4 h of 40 [micro]g/ml THH treatment. Our results suggested that THH alkaloids induced apoptosis of HL-60 cells though c-myc and NF-[kappa]B signaling pathways. This study can help to understand the molecular mechanisms involved in TH clinical function.
Table 1. Effects of THH alkaloids on HL-60 cell cycle and apoptosis.

Treatment Distribution of Apoptosis
concentration cell cycle (%) rate (%)
([micro]g/ml) [G.sub.0]/[G.sub.1] S [G.sub.2]/M

 0 40.1 38.6 21.3 0.2
25 44.8 32.4 22.8 3.1
40 52.7 24.1 23.2 9.3

Table 2. List of differentially expressed genes in HL-60 cells upon THH
treatment.

Category Description of gene Accession Fold
 Number

Apoptosis Nuclear factor of kappa light
 polypeptide gene enhance in B-cells
 inhibitor, beta (NFKBIB) AI935157 4.35
 Proteoglycan 1, secretory granule
 (PRG1) NM_002727 3.76
 Caspase 8, apoptosis-related
 cysteine protease BG529842 3.15
 Solute carrier family 25
 (mitochondrial carrier; phosphate
 carrier), member 3 BG491863 3.08
 Caspase 3, apoptosis-related
 cysteine protease AU125557 2.86
 Beta-2-microglobulin (BM) AV710740 2.72
 ATP synthase, H+ transporting,
 mitochondrial F0 complex, subunit g AV714814 2.63
 Mitochondrial ribosomal protein S12 AF058761 2.56
 Non-metastatic cells 5, protein
 expressed in (nucleoside-diphosphate
 kinase) AL043778 0.47
 C-myc binding protein AL561551 0.33
Cell cycle and HIR histone cell cycle regulation
differentiation defective homolog A (S, cerevisiae) NM_003325 2.18
 Ribosomal protein L31 AW973154 2.56
 Interleukin 10 receptor, beta BC001903 0.48
 Cyclin B2 N87720 0.32
Stress response Heat shock 70kD protein 4 BE742483 2.05
 Heat shock 90kD protein 1, beta BG336532 2.13


Acknowledgement

This work is supported by City University of Hong Kong through a Applied Research Grant (CityU Project No. 9630002).

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W.-J. Zhuang (1), C.-C. Fong (1), J. Cao (2), L. Ao (2), C.-H. Leung (1), H.-Y. Cheung (1), P.-G. Xiao (3), W.-F. Fong (1), and M.-S. Yang (1)

(1) Department of Biology & Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China

(2) Molecular Toxicology Laboratory, Third Military Medical University, Chongqing, China

(3) Institute of Medicinal Plants, Chinese Academy of Medical Science, Beijing, China

Address

W.-F. Fong. Department of Biology & Chemistry, City University of Hong Kong, Kowloon, Hong Kong, China

Tel.: ++852 2788 8031; Fax: ++852 2788 7406; e-mail: bhwffong@cityu.edu.hk
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Author:Zhuang, W.-J.; Fong, C.-C.; Cao, J.; Ao, L.; Leung, C.-H.; Cheung, H.-Y.; Xiao, P.-G.; Fong, W.-F.;
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Apr 1, 2004
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