Paclitaxel induces apoptosis via caspase-3 activation in human osteogenic sarcoma cells (U-2 OS)

ELSEVIER

Journal of Orthopaedic Research

Journal of Orthopaedic Research 23 (2005) 988-994

www.elsevier.com/locate/orthres

Paclitaxel induces apoptosis via caspase-3 activation in human osteogenic sarcoma cells (U-2 0s) K.-H. Lu

K.-H. Lue b, M.-C. Chou

b,

J.-G. Chung ',*

Department of Orthopaedic Surgery, Chung Shan Medical University Hospital, No. 110. Section I , Chien-Kuo N . Road, Taichung 402, Taiwan, ROC Institute of Medicine. Chung Shan Medical University, No. 110, Section I , Chien-Kuo N. Road, Taichung 402. Taiwan, ROC ' Department of Microbiology, China Medical University, No. 91, Hsueh-Shih Road, Taichung 404. Taiwan, ROC a

Accepted 28 January 2005

Abstract Paclitaxel has been found to exhibit cytotoxic and antitumor activity. There is little information regarding the mechanisms of apoptotic-inducing effect of paclitaxel on human osteogenic sarcoma U-2 0s cells. Several key regulatory proteins are involved in the initiation of apoptosis. Caspase-3 plays a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. We examined the effect of paclitaxel on the cell cycle arrest and apoptosis in U-2 0s cells using flow cytometric analysis and Western blotting. We also measured the inhibition of paclitaxel-induced apoptosis and the caspase-3 activity by the broad-spectrum caspase inhibitor z-VAD-fmk on U-2 0s cells. The increased levels of casapse-3 were also confirmed by cDNA microarray. Our observations were: (1) paclitaxel treatment resulted in G2/M-cycle arrest in U-2 0s cells; (2) time and dose dependent apoptosis of U-2 0s cells was induced by paclitaxel; (3) in U-2 0s cells, z-VAD-fmk blocked the paclitaxel-induced apoptosis and caspase-3 activation. These results suggest that paclitaxel-induced G2IM-cycle arrest of the G2/M phase and apoptosis via a caspase-3 pathway in U-2 0s cells. 0 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. Keywords: Paclitaxel; Osteogenic sarcoma U-2

0s cell; Cell cycle; Apoptosis; Caspase-3

Introduction

Osteosarcoma is the most common primary malignant tumor of bone, especially in children. The principles of treatment of osteosarcoma have undergone dramatic changes in the past 20 years. Until recently, 5-year survival of 20% with surgical treatment alone was considered acceptable. This outcome suggested that 80% of the patients had pulmonary metastasis (perhaps undetectable) at the time of presentation [34]. Hence, the chemotherapy is usually employed in the adjuvant situ-

* Corresponding author. Tel.: +886 4 2205 3366; fax: +886 4 2205 3764. E-mail address; [email protected] (J.-G. Chung).

ation to improve the prognosis and long-term survival. However, their values were limited by toxicity or lack of efficacy. Various inducers of apoptosis and the mechanisms of apoptosis have been reported [11,26]. Many chemotherapeutic drugs induce cancer cell death through apoptotic pathways [4,28,30]. Apoptosis is a programmed cell death involved in many physiological and pathological processes and has been widely documented to occur in a number of cell types [3,12]. It is typically accompanied by the activation of a class of death proteases (caspases) and wide-spread biochemical and morphological changes to the cell [21,24]. These changes involve chromatin condensation, shrinkage of the cell, DNA fragmentation, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighboring cells [16].

0736-0266/$ - see front matter 0 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. doi: 10.1016/j.orthres.2005.01.018

K.-H. Lu et al. I Journal qf Orthopaedic Research 23 (2005) 988-994

Paclitaxel (Taxol) was isolated from the bark of the Pacific yew Taxus brevifolia and was reported to have cytotoxic activity against a broad range of murine leukemia and solid tumors [8,33]. Those solid tumors include chemotherapy-resistant epithelial ovarian cancer, advanced breast cancer, small cell and non-small cell lung cancer, and head and neck cancer [33]. In vitro data, caspase-3 inhibitors reduced human osteoblastic Saos-2 cells from paclitaxel-induced apoptosis [25]. Human osteogenic sarcoma U-2 0s cells increased caspase-3 activity after paclitaxel exposure [lo]. We had also demonstrated that paclitaxel inhibited N-acetyltransferase activity, DNA adduct formation and gene expression in U-2 0s cells [19]. Thus far, little is known regarding the mechanisms of apoptotic-inducing effect of paclitaxel on U-2 0s cells [14,15]. Although paclitaxel interacts with microtubules and induces apoptosis in various tumor cells, whether it can induce apoptosis in U-2 0s cells is not very clear [15]. Apoptosis signal transduction and execution require the action of the cascade of caspases [2,29]. Human caspases-1 to -10 have described that activation of caspase cascade is involved in chemicals- and agentsinduced apoptosis [5]. Caspase-3 is an executioner caspase and relies on the action of the initiator caspases including caspase-8 and caspase-9 for its action [6]. However, there is no available information to address the effects of paclitaxel on the cell cycle of U-2 0 s cells and caspase-3 on paclitaxel-induced apoptosis in human U-2 0s cells. Therefore, the purpose of this study was to investigate the effects and possible mechanism of paclitaxel on the cell cycle progression and regulatory molecules of U-2 0s cells.

Materials and methods Chemicals and reagents

Paclitaxel (Taxol) was obtained from Bristol Caribbean, Inc. (NJ, USA). RPMI 1640 tissue culture medium, trypan blue, Tris-HCI, Triton X-100, propidium iodide and ribonuclease A were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), potassium phosphates and TE buffer were purchased from Merck Co. (Darmstadt, Germany). Fetal bovine serum, penicillinstreptomycin, trypsin-EDTA and glutamine were obtained from Gibco BRL (Grand Island, NY, USA). All chemicals used were reagent grade.

Human osteogenic sarcoma cell line (U-2 0s)

The human osteogenic sarcoma cell line (U-2 0s: human; female; 15 years old) was obtained from the Food Industry Research and

Development Institute (Hsinchu, Taiwan). The cells have been cultured for four generations in our laboratory and continuously checked for viability. The cells were placed into 75-cm2tissue culture flasks and grown at 37 "C under a humidified 5% C 0 2 atmosphere in RPMI 1640 medium (Sigma Chemical Company) supplemented with 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA), penicillin (100 U/ml) and streptomycin (10 mglml).

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Puclitaxel treatment

U-2 0s cells were treated with different concentrations of paclitaxel (0,0.5, I , 5,25 and 50 pM) for 6, 12, 18,24,48 and 72 h. Paclitaxel was

dissolved in DSMO, and the final concentration of the vehicle was ~ 0 . 1 % . We only added DMSO (solvent) to the control regimen (0 pM of paclitaxel). Cell morphology Cells were plated in 6-well plates (34mm) at a density of 1 x lo6 cells/well and grown for 24 h to attach to the surface of the plates completely. They were added to different concentrations of paclitaxel (0,0.5, 1,5,25 and 50 pM) and grew at 37 "C in a humidified 5%

COz for different periods of time (12, 24, 48 and 72 h). For cell morphology experiment, the culture plates were examined and photographed by phase microscopy. Cell viability assay

Cells were plated in 6-well plates (34 mm) at a density of 1 x lo6 cellslwell and grown for 24 h. They were then added to different concentrations of paclitaxel (0, 0.5, 1, 5, 25 and 50 pM). Cells were

grown at 37 "C in a humidified 5% COz for different periods of time (6, 12, 18, 24, 48 and 72 h). For cell viability experiment, the trypan blue exclusion protocol was used. Briefly, 10 pI of cell suspensions in RPMI 1640 medium were mixed with 40 pl of trypan blue, and the numbers of stained (dead cells) and unstained cells (live cells) were counted using a hemocytometer [23]. The results were rechecked by flow cytometry.

Flow cytometric analysis To estimate the proportion of U-2 0 s cells in different phases of cell cycle affected by paclitaxel, cellular DNA contents and apoptosis were measured by flow cytometry as described by Ormerod [22]. Briefly, about 1 x 106cells/well were fixed gently (drop by drop) by putting 70"h ethanol (in PBS) in ice overnight and were then resuspended in PBS containing 40 pglml propidium iodide (PI) and 0.1 mglml RNase (Sigma, St. Louis, MO, USA). After 30 min at 37 "C, 1 x lo6 cells were analyzed on a flow cytometry (Becton-Dickinson, San Jose, CA, USA) equipped with an argon ion laser at 488 nm wavelength. Then the cell cycle was determined. U-2 0 s cells were also pretreated with or without the cell permeable broad-spectrum caspase inhibitor z-VAD-fmk 3 h before paclitaxel treatment. Apoptosis of U-2 0s cells was measured by flow cytometry. Protein extraction

For Western blot analysis, U-2 0s cells were treated with different concentrations of paclitaxel (0, 0.5, 1, 5, 25 and 50 pM) for 48 h. Collected cells were washed with cold PBS. One milliliter of lysis buffer (62.5 mM Tris-HC1, 2% SDS, 10%1glycerol, 0.1% bromophenol blue) was added and the proteins were extracted. Protein content was quantitated using Bradford assay (BioRad, Hercules, CA, USA). Western blot analysis

Caspase-3 protein expression was measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot as described by Towbin et al. [31]. In all analyses, protein concentration was standardized among samples. Briefly, aliquots of cell lysates containing 50 pg were separated by 10% SDS-PAGE. Electrophoresis was carried out at 20 V/cm for 120 min. The proteins in the SDS-polyacrylamie gel were transferred onto nitrocellulose. After washing the blotted nitrocellulose twice with water, membranes were blocked in freshly prepared PBS containing 5%.

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c D N A microarray

The total RNA was extracted from U-2 0s cells treated with or without 25 pM paclitaxel for 24 h by using Qiagen RNeasy Mini Kit. The total RNA were used for cDNA synthesis and labeling, then microarray hybridization, following by flour-labeled cDNA hybridization to their complements on the chip, and the resulting localized concentrations of fluorescent molecules were detected and quantitated [ABC (Asia BioInnovations Corporation) Human UniversoChip 8k-1 x YSample Labeling/Hybridization/Chip].

DTT, 0.1YO3-[3-chloamidopropyl] dimethylammonion) 1-propanesulfonate. Fluorescence was determined after 2 h (excitation wavelength, 400 nm; emission wavelength, 505 nm) with a fluorescence plate reader (Fluoroskan Ascent; Labsystems). Statistical analysis

Statistical calculations of the data were performed using an unpaired Student's i-test and ANOVA analysis. Statistical significance was set at p < 0.05.

Caspase activity determinaiion

Results About 5 x lo6 cells were lysed in lysis buffer (1% Triton X-100,0.32 M sucrose, 5 mM EDTA, 10 mM Tris-HCI, pH 8.0, 2 mM dithiothreitol, 1 mM PMSF, 1 pg/ml aprotinin, 1 mg/ml leupeptin) for 30 min at 4 "C followed by centrifugation at 10,000x g for 30 min. For caspase activity determination, 50 p1 reaction mixtures with fluorogenic report substrate peptides specific for caspase-3. The substrate peptide (200 pM) was incubated at 37 "C with cytosolic extracts (15 pg of total protein) in reaction buffer (100mM HEPES, 10% sucrose, 10mM

EfSects of paclitaxel on cell morphology of U-2 0s cell line

Morphological changes were substantially observed in the U-2 0s cells at 72 h after the addition of

Control

0.5 pM

1 PM

25 pM

50 pM

Fig. 1. The morphology of U-2 0s cells after exposure to the different doses of paclitaxel for 72 h then were examined and photographed by phase microscope ( 2 0 0 ~ )The . cell damage apparently appeared after treatment with 5-50 pM of paclitaxel, comparing with the control plate. Control, 0.5, I , 5, 25 and 50 pM.

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paclitaxel at various concentrations were examined and photographed by light-phase microscope. It showed damaged cells which had become round and shrunken, while the control U-2 0s cells were well spread (Fig. 1). The cell damage apparently appeared after treatment with 5-50 pM of paclitaxel. Therefore, we used 25 pM of paclitaxel for further studies. Paclitaxel induced U-2 0s cell cycle arrest in G2/M phase

Cells were exposed to 25 pM paclitaxel for different time periods, the cell number percentage in each phase (GO/Gl, S and G2/M) of the cell cycle was calculated by flow cytometric analysis (Figs. 2 and 3). Paclitaxel induced the inhibition of cell growth and cell cycle progression mainly by inducing arrest of the G2/M phase in U-2 0s cells and its effect was in a time-dependent manner (GO/Gl: F = 15.779, p < 0.001; S : F = 18.702, p < 0.001; G2/M: F = 181.945, p < 0.001.). Paclitaxel induced apoptosis in U-2 0s cells

The paclitaxel treated cells showed a typical pattern of DNA content that reflected GO/Gl-, S- and G2/Mphase of the cell cycle together with a sub-GO/Gl-phase (corresponding to apoptotic cells) that were analyzed by

a,b,c.d.e

Fig. 3. The U-2 0s cells (A) were treated with 25 pM paclitaxel (B) for 24 h, washed and then harvested. The cells were fixed and stained with propidium iodide and the DNA content was analyzed by flow cytometry (FACS).

0

20

40 Time (h)

60

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Fig. 2. Effects of paclitaxel on the DNA content of U-2 0s cells. Cells were exposed to 25 pM paclitaxel for the indicated time, washed and then harvested. The cells were fixed and stained with propidium iodide and the DNA content was analyzed by flow cytometry (FACS). The cell number percentage in each phase (GOIGi, S, and G2/M) of the cell cycle was calculated and expressed. Paclitaxel induced the inhibition of cell growth and cell cycle progression mainly by inducing arrest of the G2/M phase in U-2 0s cells. The results are expressed as mean f SD of six determinations. ANOVA analysis with Scheffe posteriori comparison was used. GO/GI: F = 15.779, p < 0.001; S : F = 18.702, p < 0.001; GUM: F = 181.945, p < 0.001. aSignificantly different, p < 0.05, when compared to 0 h. bSignificantly different, p < 0.05, when compared to 6 h. ‘Significantly different, p < 0.05, when compared to 12 h. dSignificantly different, p < 0.05, when compared to 18 h. eSignificantly different,p < 0.05, when compared to 24 h.

flow cytometry (Fig. 3). The higher concentration of paclitaxel that was treated, the higher percent of apoptosis was detected in examined U-2 0s cells (Fig. 4A) (F= 98.551, p < 0.001). The effects were also in a timedependent manner (Fig. 4B) (F=63.101, p < 0.001). Paclitaxel decreased caspas-3 expressions in 17-2 0s cells

The results of Western blot analysis of caspase-3 in U-2 0s cells with or without treatment of paclitaxel are shown in Fig. 5A. The higher concentrations of paclitaxel were treated, the more expressions of caspase-3 were examined. The representative result on cDNA microarray is presented in Fig. 5B. The circle marked on the block of cDNA microarray indicated that the caspase-3 gene is up regulated.

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(A)

C

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5

25

50 pM

Caspase-3

(A)

Concentrations of paclitaxel (pM)

Caspase-3 0

(B)

20

40

60

80

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Fig. 4. (A) Dose-dependent cell death of U-2 0s cells induced by paclitaxel. The cells were treated with 0, 0.5, 1, 5, 25 and 50 pM paclitaxel for 24 h. The results are expressed as mean ? SD of six determinations. ANOVA analysis with Scheffe posteriori comparison was used. F = 98.551,~< 0.001. aSignificantly different,p < 0.05, when compared to 0 pM. bSignificantly different, p < 0.05, when compared to 0.5 pM. 'Significantly different, p < 0.05, when compared to 1 pM. dSignificantly different, p < 0.05, when compared to 5 pM. 'Significantly different, p
Inhibition of paclitaxel-induced apoptosis by the caspase-3 inhibitor z-VAD-fmk on U-2 0s cells

In order to examine whether caspase-3 activation is involved in the apoptosis triggered by paclitaxel, U-2 0s cells were pretreated with or without the broad-spectrum caspase inhibitor z-VAD-fmk 3 h before paclitaxel treatment. After treatment with z-VAD-fmk and paclitaxel, inhibition of paclitaxel-mediated caspase-3 activation (12 h: p < 0.05; 24 h: p <0.001; 48 h: p < 0.01; 72 h: p < 0.001) was accompanied by the marked attenuation of paclitaxel-induced apoptotic cell death (12 h: p < 0.001; 24 h: p < 0.001; 48 h: p < 0.001; 72 h:

Fig. 5. (A) Western blot analysis of caspase-3 in 0.5, 1, 5, 25 and 50 pM paclitaxel treated U-2 0s cells for 48 h. Dose-dependent increase of caspase-3 activation is evident. (B) The cDNA microarray was produced from U-2 0s cells treated with 25pM paclitaxel as described in Materials and Methods. The total RNA was extracted, prepared for cDNA then cDNA hybridization, and made gene regulation on the chip. Red color spot is up-regulation and green color spot is down-regulation. Circle mark (caspase-3) is the gene upregulation. (For interpretation of the references of color in this figure legend, the reader is referred to the web version of this article.)

p < 0.001) (Fig. 6 ) . The results also indicate that activation of caspase-3 contributes to paclitaxel-induced apoptosis.

Discussion Osteosarcoma accounts for approximately 20-22% of all primary sarcoma of bone, excluding the marrowbased malignancies such as myeloma, lymphoma, and leukemia and it is a tumor with a poor prognosis in the absence of effective chemotherapy. Conventional osteosarcoma belongs to the category of the most aggressive and highly lethal osseous neoplasm [20]. Before the advent of chemotherapy, treatment by surgery alone resulted in low 5-year survival rates within the range of 10-20% [9]. The general treatment plan consists of preoperative (neoadjuvant) chemotherapy and sometimes irradiation followed by unresectable spine lesions and limb-sparing surgical excision (if possible) and a postoperative chemotherapeutic regimen modified according to the extent of tumor necrosis.

K.-H. Lu et al. I Journal of Orthopaedic Research 23 (2005) 988-994

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pM paclitaxel

+U2 + 25 pM paditaxel+ z-VAD-FMK

50

T

*** P

***

"

0

(B)

80

Time (h)

20

40

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80

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Fig. 6. (A) Time-dependent inhibition of paclitaxel induced caspase-3 activation by z-VAD-fmk. The U-2 0s cells were treated with 25 pM paclitaxel and with or without z-VAD-fmk pretreatment before examined at different time (0, 12, 24, 48 and 72 h). The results are expressed as mean f SD of six determinations. (B) Tme-dependent inhibition of paclitaxel induced apoptosis by z-VAD-fmk. The U-2 0s cells were treated with 25 pM paclitaxel and with or without z-VADfmk pretreatment before examined at different time (0, 12, 24, 48 and 72 h). The results are expressed as mean f SD of six determinations. Student's t-test was used for with and without z-VAD-fmk treated groups. ' p < 0.05, " p < 0.01, "'p < 0.001.

Cell deaths can be divided into apoptosis and necrosis. The major difference between both is the active participation of the cells in the process [36]. The signal transduction pathway lead to apoptosis which is a highly organized physiological mechanism for destroying injured and abnormal cells [35] and may play an important role in some cellular processes of cell cycle. Some of the anticancer drugs, which are able to intercalate with DNA in cancer cells after applying at optimal doses in vivo, do arrest cancer cells in the cell cycle [I]. Furthermore, mitochondrial cytochrome C releases an important control point in caspase activation and apoptosis [7]. It is suggested that the susceptibility to apoptosis-inducing effects of chemotherapeutic drugs may depend on the intrinsic ability of tumor cells to respond by apoptosis [32,36]. We used flow cytometry assay for cell cycle and apoptosis, and the experimental data presented here confirm that paclitaxel could interfere with the cell cycle at

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the G2/M phase and induce apoptosis in U-2 0s cells in a dose- and time-dependent manner. Caspase-3 plays a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. In an attempt to identify the pathway of apoptosis in U-2 0s cells in response to paclitaxel, caspase-3 activation was investigated. The results from Figs. 5 and 6 show that paclitaxel could induce caspase-3 activation as detected by the decrease of the paclitaxelinduced caspase-3 activities after treatment with z-VAD-fmk. The increased levels of caspase-3 were also confirmed by cDNA microarray (Fig. 5B). Other investigators have reported that the ratio between pro- and anti-apoptotic proteins determines in part the susceptibility of cells to a death signal [13,37]. The activation of caspase-3 induced PARP cleavage, chromosomal DNA break and finally the occurrence of apoptosis [27]. A broad-spectrum caspase inhibitor z-VAD-fmk, which has no effect on paclitaxel-induced increase in cytochrome C, but inhibits the ensuing caspase-3 cleavage and activity [17,18], blocked the apoptosis in this study, suggesting that caspase-3 might have a critical role in the apoptosis induced by paclitaxel. In this study, it is clear that paclitaxel exerts apoptotic effects in U-2 0s cells by acting on caspase-3. Paclitaxel initiates activation of caspases and in turn the caspase activation leads to apoptotic cell death. Still, the mechanism whereby paclitaxel activates caspases on U-2 0s cells is presently unknown. We only demonstrated that paclitaxel triggered apoptosis through activation of caspase-3. Our results should help in further understanding the paclitaxel-induced apoptosis in U-2 0s cells, which is important since paclitaxel has a potential role in therapies of human cancer in the future. These results provide an interesting correlation between caspase-3 activity and paclitaxel-induced apoptosis, and deserve to be pursued further. In this study, our results show: (1) in vitro paclitaxel treatment resulted in G2/M-cycle arrest in U-2 0s cells; (2) time and dose dependent apoptosis of U-2 0s cells was induced by paclitaxel; (3) in U-2 0s cells, z-VADfmk blocked the paclitaxel-induced apoptosis and caspase-3 activation. In conclusion we have demonstrated that paclitaxel induce apoptosis in U-2 0s cells by activating caspase-3, leading to G2/M arrest. Future studies focusing on cell signaling and biological significance of paclitaxel-induced apoptosis would lead to explore the mechanisms of chemotherapeutic potency of paclitaxel in human osteogenic sarcoma. Acknowledgement

This work was supported by grant CSMU 90-OM-B014 from the Research Section of Chung Shan Medical University.

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