• E-mail
• Print
• Comment
• Font Size
• Digg
• del.icio.us
• Discuss article

Gemcitabine Inhibits Viability, Growth, and Metastasis of Osteosarcoma Cell Lines

Posted on: Sunday, 7 August 2005, 03:01 CDT

Abstract

Gemcitabine (dFdCyd) is an analog of cytosine arabinoside with anti-tumor activity in several human cancers. However, the efficacy of this compound in osteosarcoma has not been fully elucidated. Here we assessed the anti-tumor activity of gemcitabine using osteosarcome cell lines. In 9 human osteosarcoma cell lines (G292, HOS, MG63, NY, SaOS, HuO, HuO-3N1, HuO9, HuO9-N2), gemcitabine at the doses of >100 nM showed significant cytotoxicity. In HOS and MG63 cell lines, gemcitabine inhibited DNA synthesis as determined by IdU labeling assay and induced apoptosis as determined by DNA fragmentation assay and May-Giemsa staining. In C3H mice inoculated s.c. with a murine osteosarcoma cell line, LM8, treatment of the mice with gemcitabine showed reduced size of the primary tumor associated with increased apoptotic cells and a virtual absence of metastatic lesions in the lung. Gemcitabine thus had anti-tumor activity on osteosarcoma cell lines both in vitro and in vivo. The result would provide a cellular basis for application of gemcitabine to patients with osteosarcoma.

2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Keywords: Gemcitabine: Osteosarcoma; Apoptosis; Metastasis

Introduction

Osteosarcoma is a mesenchymally derived, high-grade bone sarcoma that is the most frequently diagnosed primary malignant bone tumor with high metastatic rate to the lung. The use of aggressive combination of chemotherapy plus surgery for osteosarcoma results in a 2-year metastasis-free survival rate of 60-65% although this rate has not changed over the past 15 years despite alterations in the chemotherapeutic regimens used [2,9,12,16].

Gemcitabinc (dFdCyd) is an analog of cytosine arabinoside which shows good anti-tumor activity in a variety of solid tumors, including experimental solid tumors, non-small cell lung cancer, ovarian, head and neck, and pancreatic carcinomas [4,6,10,11,15]. Gemcitabine is metabolized intracellulary by nucleotide kinases to the active diphosphate and triphosphate nucleoside and the cytotoxic effect of this agent is attributed to a combination of the two actions of these nucleosides which inhibits DNA synthesis and cell proliferation [15]. In addition, gemcitabine, like other chemotherapeutic agents, was reported to have capacity to induce apoptosis in cancer cells [3].

Although recent preclinical and clinical studies have shown that gemcitabine has some activity against xenografts of sarcoma cell lines [5] and in patients with advanced sarcomas [7,13], the efficacy of gemcitabine in osteosarcoma has not been fully addressed so far when compared with other solid tumors. Gemcitabine has received Food and Drug Administration of USA (FDA) approval for pancreatic cancer in 1996, non-small-cell lung cancer in 1998, breast cancer in 2004. Thus, we here-in investigated the effect of gemcitabine on the viability, DNA synthesis, apoptosis, and in vivo growth and metastasis of osteosarcoma cell lines derived from humans and mice. Our findings indicate that gemcitabine has anti-tumor activity on osteosarcoma cell lines and suggest that gemcitabine may be useful for treatment of human osteosarcoma.

Materials and methods

Reagents

Gemcitabine was purchased from Eli Lilly (Indianapolis, IN).

Cell culture

All the cell lines used in this study were obtained from the American Type Culture Collection (Rockville, MD) except for a murine osteosarcoma cell line LM8 that was purchased from Riken Cell Bank (Tsukuba, Japan), human dermal fibroblast was purchased from Cell Applications, Inc. Human osteosarcoma cell lines. G292, HOS, MG63, NY, SaOS, HuO, HuO-3N1, HuO9, HuO9-N2, were maintained in RPMI1640 (Gibco/Invitrogen, Carsbad, CA) with 10% FCS. Human pancreatic cancer cell line MIA PaCa-2 was cultured in DMEM (Gibco/Invitrogen. Carsbad, CA) with 10% FCS. Human dermal fibroblast was culture in Fibroblast Basal Medium.

Cell viability assay

1 10^sup 4^ cells were cultured with or without gemcitabine for 72 h. Crystal violet assay: Cells were stained with 50 l crystal violet solution (0.1%) for 10 min, rinsed 10 times in tap water, and the plates were dried on paper. After 1 h, 100 l of equal volume mixtures of ethanol and 0.1 M sodium phosphate were added and the absorbance at 570 nm was measured. WST assay: The cell viability was then determined by WST assay using Tetra Color ONE kit (Seikagaku Corporation, Tokyo, Japan) according to the manufacturer's instructions.

IdU labeling

1 10^sup 4^ cells were cultured with or without gemcitabine for 12 h. IdU labeling assay was then performed using DNA IdU labeling and detection kit (Takara Biomedical, Osaka, Japan) according to the manufacturer's instruction.

DNA fragmentation

3 10^sup 5^ cells were cultured with or without 1 M gemcitabine. DNA samples were prepared by using ApoLadder EX kit (Takara Biomedical, Osaka, Japan) according to the manufacturer's instruction and loaded onto 2% agarose gel (10 g/lane). Electrophoresis was run under 100 V. DNA fragmentation was visualized after staining with ethidium bromide by transillumination with UV light.

Light microscopy

HOS cells or MG63 cells were cultured with or without 1 M gemcitabine for 24 h or 48 h, respectively, and then fixed and stained with Giemsa solution. Morphological changes were visualized by light microscopy.

Transplants/metastasis of LM8 tumor cells in mice

A murine osteosarcoma cell line, LM8 cells [1], (1 10^sup 7^ cells/ mouse) in 0.2 ml EMEM were injected s.c. into the back of male C3H mice aged at 5 weeks (CLEA Japan, Shizuoka, Japan) on day 0. On day 7, 14, and 21, 150 mg/kg of gemcitabine or vehicle only was given i.p. to 7 mice per group. The mice were weighed once every other day and the size of the primary tumors were evaluated weekly. On day 28, all mice were sacrificed and primary tumors were resected for HE and TUNEL staining that using in situ TUNEL staining kit (Takara Biomedicals, Osaka, Japan) according to the manufacturer's instruction. Metastic nodules on surface of lungs were counted with a low-powered stereomicroscope.

Data analysis

Data are summarized as mean SD. Statistical analysis was performed using the unpaired Student t-test. p < 0.05 was considered to be significant.

Results

Cytotoxic effect of gemcitabine on human osteosarcoma cell lines

To investigate the cytotoxicity of gemcitabine in osteosarcoma, we examined the effect of various doses of gemcitabine on the viability of human osteosarcoma cell lines using crystal violet assay. As shown in Fig. 1A and B, gemcitabine at the dosage of 100 nM began to cause cell death, and the dosage of 5 M appeared to reach maximum effect in all the human osteosarcoma cell lines examined (G292, HOS, MG63, NY, SaOS, HuO, HuO-3N1, HuO9, HuO9-N2). The efficacy of gemcitabine in human osteosarcoma cell lines was largely equivalent to that of gemcitabine in MIA PaCa-2, a pancreatic cancer cell line that was sensitive to gemcitabine [3].

Inhibition of DNA synthesis and increased apoptosis by gemcitabine

To explore the mechanisms underlying the gemcitabine-induced cytotoxicity in human osteosarcoma cell lines, we examined the effect of gemcitabine on DNA synthesis and apoptosis using two representative human osteosarcoma cell lines (HOS and MG63). Gemcitabine inhibited incorporation of IdU into genomic DNA in HOS (Fig. 2A) and MG63 (Fig. 2B) cell lines in a dose-dependent manner. Gemcitabine also induced DNA fragmentation (Fig. 3A) and morphological changes such as nuclear fragmentation and apoptotic bodies (Fig. 3B) in HOS and MG63 cells, which were characteristic features of cells undergoing apoptosis.

Fig. 1. Cytotoxic effect of gemcitabine on human osteosarcoma cell lines: 9 human osteosarcoma cell lines (G292, HOS, MG63, NY, SaOS, HuO, HuO-SN1, HuO9, HuO9-N2) and 1 human pancreatic cancer cell line (MIA PaCa-2) were cultured in the presence of varying doses of gemcitabine for 72 h. Viable cells were then assessed by using crystal violet assay. p values were under 0.05 over >100 nM doses of gemcitabine.

Fig. 2. Inhibition of DNA synthesis by gemcitabine: HOS (A) or MG63 (B) cells were cultured in the presence of varying doses of gemcitabine for 12 h. Then, IdU labeling assay was performed as described in the Materials and methods. Data are indicated as the mean SD. *p < 0.05, significantly different from the mean value of the corresponding control response. Similar results were obtained in three repeated experiments.

Decreased primary tumor size and lung metastasis in gemcitabine- trealed mice

A murine osteosarcoma cell line (LM8) was shown to be transplantable to C3H mice and develop tumors with high metastatic potential to the lung after inoculation into the skin [1]. We assessed anti-tumor activity of gemcitabine using this model. After s.c. inoculation of LM8 cells (1 10^sup 7^ cells), LM8 tumors exhibited rapid growth and reached a size of about 10 cm^sup 2^ within 4 weeks (Fig. 4A and B). Multiple metastatic nodules were found grossly or histologically in the lungs of LM8-inoculated mice 4 weeks after inoculation (Fig. 5 and data not shown). Treatment of the mice with 150 mg/kg of gemcitabine given once weekly after inoculation of LM8 showed significant reduction of the primary tumor size associated with increased apoptotic cells inthe tumor as judged by TUNEL staining (Fig. 4C). Gemcitabine-treated mice also showed a virtual absence of metastatic lesions in the lung (Fig. 5A and B). We found there was little difference in lung and liver histology (Fig. 5C) and in the weight (data not shown) of the mice treated or untreated with gemcitabine and no obvious abnormality in the treated mice at this dosage.

Control study

As control study, we examined the effect of various doses of gemcitabine on normal human fibroblasts with HOS and MG63 using WST assay (Fig. 6A). Cytotoxicity of gemcitabine for normal human fibroblast was similar to HOS and MG63. In addition, the efficacy of gemcitabine in HOS (Fig. 6B) and MG63 (Fig. 6C) was largely equivalent to that of other anti-tumor drugs (doxirbicin, ifosfamide, and methotrexate) currently in use in the management of osteosarcoma [2].

Discussion

In this study, we demonstrated that gemcitabine had cytotoxicity (Fig. 1), inhibited DNA synthesis (Fig. 2), induced apoptosis (Fig. 3), and suppressed the growth of primary tumors (Fig. 4) and pulmonary metastasis (Fig. 5) in osteosarcoma cell lines.

Fig. 3. Induction of apoptosis by gemcilabine: (A) DNA fragmentation analysis. HOS (left panels) or MG63 cells (right panels) were treated (+) or untreated (-) with 1 M gemcitabine for indicated times. Then nuclear DNA was prepared as described in the Materials and methods and samples were electrophoresed on 2% agarose gel. Representative pictures of three repeated experiments with similar results were shown. (B) Giemsa staining for detection of apoptotic cells. HOS (left panels) or MG63 cells (right panels) were treated (+) or untreated (-) with gemcitabine for 24 h or 48 h, respectively, and then fixed and stained with Giemsa solution. Morphological changes were visualized by light microscopy. Representative pictures of three repeated experiments with similar results were shown.

Fig. 4. Inhibition of the development of osteosarcoma by gemcitabine: LM8 cells were inoculated s.c. into the back of C3H mice on day 0. The mice were treated i.p. with 150 mg/kg of gemcitabine or vehicle on day 7, 14, and 21. (A) Representative pictures showing gross appearance of the primary tumor treated (+) or untreated (-) with gemcitabine on day 28. (B) The size of the primary tumor evaluated weekly. (C) HE (left panels) and TUNEL (right panels) staining of the sections of the primary tumor treated (+) or untreated (-) with gemcitabine on day 28. Data are indicated as the mean SD (n = 7). *p < 0.05, significantly different from the mean value of the corresponding control response. Similar results were obtained in three repeated experiments.

Importantly, gemcitabine showed significant cytotoxicity even at dose of 100 nM in human osteosarcoma cell lines and the cytotoxicity was almost equivalent to that observed in MIA PaCa-2, that was sensitive to gemcitabine [3] (Fig. 1). These results suggest that gemcitabine may have therapeutic potential for osteosarcoma as well as other human cancers [8,14].

Gemcitabine inhibited DNA synthesis and induced apoptosis in HOS and MG63 cell lines (Figs. 2 and 3). These effects of gemcitabine on cancer cells have been described previously in several tumors [3,12]. Thus, cytotoxicity of gemcitabine on osteosarcoma cell lines could be attributed to inhibition of DNA synthesis or induction of apoptosis or both.

Cytotoxicity of gemcitabine was almost equivalence to other anticancer drugs (doxorubicin, ifosfamide, MTX) that commonly used in the management of osteosarcoma (Fig. 6B and C) [2]. However, gemcitabine had also cytotoxicity to normal cell (Fig. 6A), and various side effects were reported [7,10]. Therefore, unfavorable effects of gemcitabine must be carefully investigated or examined in its clinical use for treatment of osteosarcoma.

Fig. 5. Inhibition of pulmonary metastasis by gemcitabine: LM8 cells were inoculated s.c. into the back of C3H mice on day 0. The mice were treated i.p. with 150 mg/kg of gemcilabine or vehicle on day 7, 14, and 21. On day 28, lungs were removed and metastatic nodules on their surface were counted with a low-powered stereomicroscope. (A) Representative microscopic pictures of the lungs (HE staining) treated (+) or untreated (-) with gemcitabine on day 28 showing the presence of metastatic nodules. (B) Quantitative analysis of metastatic nodules in the lungs. (C) Compare lung (upper panels) and liver (lower panels) histology of normal mouse (left panels) with gemcitabine treated (same dose of (A)) mouse (right panels). Data are indicated as the mean SD (n = 7). *p < 0.05, significantly different from the mean value of the corresponding control response.

Fig. 6. Action of gemcitabine on normal human cell: (A) HOS, MG63, and normal human dermal fibroblast were cultured in the presence of varying doses of gemcitabine for 72 h. Next compare with gemcitabine and other anti-tumor agents. HOS (B) or MG63 (C) cells were cultured in the presence of varying doses of gemcitabine or doxorubicin or ifosfamide or methotrexate (MTX) for 72 h. Viable cells were then assessed by using WST assay. Similar results were obtained in three repeated experiments.

In summary, we demonstrated that gemcitabine showed anti-tumor activity both in vitro and in vivo in osteosarcoma cell lines. Our data suggest that gemcitabine may be an option for the treatment of osteosarcoma that has poor prognosis and awaits for new treatments.

Acknowledgement

We thank Dr. Tetsu Yamane, Dr. Hitoshi Ohnuma for discussion and technical assistance, Ms. Yuko Ohnuma for secretarial assistance. This work was supported in part by the grant from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

References

[1] Asai T, Ueda T, Itoh K, et al. Establishment and characterization of a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung. Int J Cancer 1998;76:418-22.

[2] Bacci G, Ferrari S, Longhi A, et al. High dose ifosfamide in combination with high dose methotrexate, adriamycin and cisplatin in the neoadjuvant treatment of extremity osteosarcoma. J Chemother 2002;14(2):198-206.

[3] Bold RJ, Chandra J, McConkey DJ. Gemcitabine-induced programmed cell death (apoptosis) of human pancreatic carcinoma is determined by Bcl-2 content. Ann Surg Oncol 1999;6:279-85.

[4] Boven E, Schipper H, Erkelens CAM, et al. The influence of the schedule and the dose of gemcitabine on the anti-tumor efficacy in experimental human cancer. Br J Cancer 1993;68:52 6.

[5] Braakjuis BJ, Ruiz van Harpere VW, Boven E, et al. Schedule- dependent antitumor effect of gemcitabine in in vivo model systems. Semin Oncol 1995;22:42-6.

[6] Braakuhuis BJM. Van Dongen GAMS, Vermorken JB, Snow GB. Preclinical in vivo activity of 2',2'-difluoro-2'-deoxycytidine (gemcitabine) against human head and neck cancer. Cancer Res 1991;51:211-4.

[7] Buesa JM, Eosa R, Fernandez A, et al. Phase I clinical trial of fixed-dose rate infusional gemcitabine and dacarbazine in the treatment of advanced soft tissue sarcoma, with assessment of gemcitabine triphosphate accumulation. Cancer 2004;101(10): 2261-9.

[8] Casper ES, Green MR, Kelsen DP, et al. Phase II trial of gemcitabine (2',2'-difluoro-2'-deoxycytidine) in patients with adenocarcinoma of the pancreas. Invest New Drugs 1994;12: 29-34.

[9] Eiber F, Chilian A, Eckardt J, et al. Adjuvant chemotherapy for osteosarcoma: a randomized prospective study. J Clin Oncol 1987:5:21-6.

[10] Gallelli L, Nardi M, Prantera T, et al. Retrospective analysis of adverse drug reactions induced by gemcitabine treatment in patients with non-small cell lung cancer. Pharmacol Res 2004;49(3):259-63.

[11] Hertel LW, Border GB. et al. Evaluation of the antitumor activity of gemcitabine (2',2'-difluoro-2'-deoxycytidine). Cancer Res 1990:50:4417-22.

[12] Link MP, Goorin AM, Miser AW, et al. The effect of adjuvant chemotherapy on relapse-free survival in patients with osteosarcoma of the extremity. New Eng J Med 1986:314: 1600-6.

[13] Merimsky O, Meller I, Flusser G, et al. Gemcitabine in soft tissue or bone sarcoma resistant to standard chemotherapy: a Phase II study. Cancer Chemother Pharmacol 2000;45:177-81.

[14] Moore M, Andersen J, Burris H, et al. A randomized trial of gemcitabine versus 5-FU as first-line therapy in advanced pancreatic cancer. Proc Am Soc Clin Oncol 1995;14:199.

[15] Plunkett W, Huang P, Xu Y, et al. Gemcitabine: metabolism, mechanisms of action, and self-potentiation. Semin Oncol 1995;22:3- 10.

[16] Skinner KS, Eliber FR, Holmes C, et al. Surgical treatment and chemotherapy for pulmonary metastasis from osteosarcoma. Arch Surg 1992;127:1065-71.

Takashi Ando(a,b,*), Jiro Ichikawa(a,b), Atsushi Okamoto(b), Kachio Tasaka(b), Atsuhito Nakao(b,c), Yoshiki Hamada(a)

a Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110, Shiiuokato, Tamaho, Yamanashi 409- 3898, Japan

b Department of Immunology, Faculty of Medicine, University of Yamanashi, Yamanashi 409-3898, Japan

c Atopy Research Center, School of Medicine, Juntendo University, Tokyo 113-8421, Japan

Accepted 21 January 2005

* Corresponding author. Address: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110, Shimokato. Tamaho, Yamanashi 409-3898, Japan. Tel./fax: +81 55 273 9542.

E-mail address: takakun@yin.or.jp (T. Ando).

Copyright Journal of Bone and Joint Surgery, Inc. Jul 2005

Source: Journal of Orthopaedic Research

More News in this Category