Triptolide exerts anti-tumor effect on oral cancer and KB cells in vitro and in vivo

Oral Oncology 45 (2009) 562–568

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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology

Triptolide exerts anti-tumor effect on oral cancer and KB cells in vitro and in vivo Yuan-Wu Chen a,b, Gu-Jiun Lin c, Wei-Tso Chia a, Chih-Kung Lin d, Yi-Ping Chuang e, Huey-Kang Sytwu a,e,f,* a

Graduate Institute of Medical Sciences, National Defense Medical Center, No. 161, Section 6, Min-Chuan East Road, Neihu 114, Taipei 114, Taiwan, ROC Department of Oral and Maxillofacial Surgery, Tri-Service General Hospital, Taiwan, ROC Graduate Institute of Life Sciences, National Defense Medical Center, Taiwan, ROC d Department of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taiwan, ROC e Department of Microbiology and Immunology, National Defense Medical Center, Taiwan, ROC f Department of Medical Research, Tri-Service General Hospital, National Defense Medical Center, Taiwan, ROC b c

a r t i c l e

i n f o

Article history: Received 11 August 2008 Received in revised form 7 October 2008 Accepted 8 October 2008 Available online 8 April 2009 Keywords: Oral squamous cell carcinoma Triptolide Apoptosis

s u m m a r y Triptolide (TPL), a diterpenoid triepoxide purified from the Chinese herb Tripterygium wilfordii Hook F, has been reported to potentiate the anti-tumor effect in various cancer cells. However, the effect of TPL on oral cancers is not yet evaluated. Herein we first demonstrate that TPL induces prominent growth inhibition and apoptosis in two oral cancer cell lines, SCC25 and OEC-M1 and in KB cells. Our results indicate that TPL induces a dose-dependent apoptosis of these cells at nanomolar concentration. Apoptosis signalings are both activated through time upon TPL treatment detected by elevated caspase-3, 8, 9 activities. In xenograft tumor mouse model, TPL injection successfully inhibits the tumor growth via apoptosis induction which was demonstrated by TUNEL assay. These results demonstrate that TPL exerts antitumor effect on oral cancer and KB cells and suggest further the potential of TPL combining with other chemotherapeutic agents or radiotherapy for advanced oral cancer. Ó 2008 Elsevier Ltd. All rights reserved.

Introduction Head and neck squamous cell carcinoma (HNSCC), including oral squamous cell carcinoma (OSCC), is the sixth most prevalent malignancy worldwide, and the third most common cancer in developing countries.1,2 The clinical outcome and prognosis of OSCC remains dismal; more than 50% of patients die of this disease or complications within 5 years.3 Because the chemotherapy effect on the end-stage of oral cancer is uniformly poor, the discovery of the potential therapeutic drugs for malignant oral tumor has been the most essential and emergent issue. Phytochemicals and herbal extracts have recently been investigated for their inhibitory ability against cancer cell growth and metastasis. These compounds are suggested to be the candidates of novel chemotherapeutic agents or adjuvants that improve the anti-cancer effect with standard treatments.4 Triptolide is a diterpenoid triepoxide derived from the herb Tripterygium wilfordii that has been used as a natural medicine in China for hundreds of years.5 Previous studies have shown that TPL exerts both immunosuppressive and anti-inflammatory activities, such as inhibition of cytokine gene expression in T cells.6,7 It has been used successfully for the treatment of rheumatoid arthritis and lupus erythematosus.8,9

Recently, TPL has been found to be reducing proliferation of a variety of cell lines and combating cancers.5,10,11 It has reported to be an effective inducer of apoptosis in solid cancer cells, including breast, prostate, and lung cancers.12 Although the mechanism is not well elucidated, it has been suggested that TPL might induce apoptosis by altering pathways involving p21 and p5313 and by inducing caspase-dependent cell death via the mitochondrial pathway in leukemia cells.14 Therefore, TPL has been used to act synergistically with conventional chemotherapeutic drugs to get more efficient inhibition on the growth and metastasis of various solid tumors.13,15 So far, there is no available information about the anti-tumor effects of TPL on human oral cancer cells. This study is to investigate the anti-cancer properties of TPL on human cell lines, KB, and oral cancer SCC25 and OEC-M1. Therapeutic effect of TPL has also been addressed in xenograft tumor bearing mouse model. It reveals that TPL exerts anti-tumor effect by apoptosis induction and may be useful for the prevention and treatment of patients with advanced oral cancer.

Materials and methods Cells and chemicals

* Corresponding author. Address: Graduate Institute of Medical Sciences, National Defense Medical Center, No. 161, Section 6, Min-Chuan East Road, Neihu 114, Taipei 114, Taiwan, ROC. Tel.: +886 2 87923100x18540; fax: +886 2 87921774. E-mail address: [email protected] (H.-K. Sytwu). 1368-8375/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2008.10.007

KB cancer cells were purchased from American Type Culture Collection (ATCC CCL-17; American Type Culture Collection,

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Rockville, MD, USA). OEC-M1 was obtained from gingival epidermoid carcinoma of a Taiwanese patient. SCC25 was obtained from tongue squamous cancer cells. These two cell lines were kindly provided by Dr. Jenn-Han Chen, Department of Dentistry, National Defense Medical Center, Taiwan. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES, and 1% penicillin/streptomycin. TPL (Calbiochem, San Diego, CA, USA), was dissolved in dimethyl sulfoxide as a stock of 10 mM and added to cells at the indicated concentrations.

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San Diego, CA, USA) as well as 5 ll propidium iodide and incubated for 15 min at room temperature in the dark according to the manufacturer’s instructions. At the end of incubation, 400 ll of binding buffer was added, and the cells were analyzed immediately by flow cytometry. Flow cytometric analysis was performed with a FACSCaliber using the CellQuest software (BDIS). The analyses of cells stained with V/PI were presented: Annexin V+/PI = apoptotic; Annexin V /PI+ = necrotic (or late stage apoptotic) and annexin V /PI+ = necrotic cells.18 Determination of caspase-3,8,9 activities

Growth inhibition assay Cells in logarithmic growth phase were cultured at a density of 10,000 cells/well in a 24-well plate. The cells were exposed to various concentrations of TPL for 72 h. The methylene blue dye assay was used to evaluate the effect of TPL on cell growth, as described previously.16 The IC50 value resulting from 50% inhibition of cell growth was calculated graphically as a comparison with control growth.17

Caspase-3 activity was measured by CaspACE Assay System Fluorometric Kit (Promega Corporation, Madison, WI, USA); and caspase-8, 9 activity was determined by caspase-8 and caspase-9 Fluorimetric Assay, respectively (R&D Systems, Minneapolis, MN, USA) Cells were initially seeded at a density of 1  106 in 100mm2 dishes. After treatment for the indicated time with various concentrations of TPL, caspase-3, 8, 9 activities were measured by the cleavage of the fluorometric substrate according to manufacturer’s instructions.

Annexin V staining Xenograft tumor model After treatment with TPL for 24 h, cells were harvested and washed twice with cold PBS and resuspended in binding buffer (10 mM HEPES/NaOH (pH 7.4), 140 mM NaCl and 2.5 mM CaCl2) at a concentration of 1  106 cells/ml. Aliquots cells were stained with 5 ll ready made Annexin V-FITC solution (BD Pharmingen,

Eight-week-old NOD/SCID (NOD.CB17 Prkdc scid/J, National Laboratory Animal Center, Taiwan) mice were maintained in microisolators under specific pathogen free condition. These mice were fed with sterile food and chlorinated sterile water. Thirty-six mice

Figure 1 TPL inhibits oral cancer and KB cell growth in vitro. (A) Morphological variations of KB, SCC25, OEC-M1 cells treated with 0, 20, 80 nM TPL for 24 h (arrows indicate dead cells). (B) Growth inhibition was measured when treated various doses of TPL with times on KB, SCC25, OEC-M1 cells at 24–96 h. (C) After 72 h treatment, the IC50 of TPL was 3.56 nM on KB, 3.75 nM on SCC25 and 4.13 nM on OEC-M1 cell. Survival proportion (%) indicates the relative value compared with control groups (*, p < 0.05).

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were divided into three groups; each group of mice was subcutaneously injected with 2  106 KB, 4  106 SCC25, and 6  106 OECM1 cells, respectively. Six mice in each group were further treated with TPL (0.15 mg/kg BW/day/i.p.) and six mice in each group were daily injected with vehicle control. TPL was first injected on day 3, before the tumor was palpated, in each group of mice, and continuously administrated until days 15, 42, and 84 in KB-, SCC25-, and OEC-M1-bearing mice, respectively. The size of the transplanted tumors was measured by gauged calipers every 3 days and the tumor volume was calculated using the formula V = 1/2  (length  width2). At the end of treatment, the mice were sacrificed, and the tumors were removed, weighed, and photographed. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay

to the tumor slides. The nucleated cells were counted using H&E counter staining under light field. The nuclei that stained as green were determined as apoptotic cells. The apoptotic index (AI) was calculated as the percentage of positive staining cells: AI = number of apoptotic cells (green)/total number of nucleated cells.34 Statistical analysis The Statistical Package for the Social Science (SPSS) for Microsoft Windows 10.0 was used to complete the analysis of the collected data. t-test, one-way analysis of variance (ANOVA) and the Scheffe post hoc test were used to determine whether any significant relationships exist among quantitative results. Values of p < 0.05 were considered significant. Results

Tumors removed from the mice were formalin fixed and paraffin-embedded. Five-micrometer thick sections were stained and examined. For in situ staining of apoptotic cells, the TUNEL method was performed using an In Situ Cell Death Detection Kit, Fluorescein (Roche, Mannheim, Germany). The staining procedures were followed by the manufacturer’s instructions. One thousand cells (1000 cells/field  10 fields) were evaluated under 400 objectives

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Growth inhibitory effect KB, SCC25 and OEC-M1 cancer cells were treated with different concentrations of TPL respectively for 24 h and the morphology was observed under a Nikon phase-contrast microscope. When treated with TPL, cell death was observed (Fig. 1A). A dose-depen-

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Figure 2 Induction of apoptosis in oral cancer and KB cell lines. (A) KB, (B) OEC-M1, (C) SCC25 cells were treated with various concentrations (20–80 nM) of TPL for 24 h, and annexin V/PI staining was performed (X axis: annexin V; Y axis: PI). Percentage value indicates the proportion of apoptotic cells (annexinV+/PI ). (D) Apoptotic cells (annexin V+/PI ) of (A)–(C) were quantified by the bar graphs. Apoptosis (%) indicates the relative value compared with control groups (*, p < 0.05).

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dent effect of TPL was revealed (Fig. 1A); less than 50% of cells survive 80 nM TPL. Evaluation of the growth inhibition effect of TPL against various human oral cancer cell lines was also conducted at various doses (0–80 nM) and time points (0–96 h) (Fig. 1B). After a 72 h treatment, the IC50 for the oral epidermoid carcinoma cell line KB was 3.56 nM, for the tongue squamous cancer cell line SCC25 it was 3.75 nM and for the gingival epidermoid carcinoma cell line OEC-M1 it was 4.13 nM (Fig. 1C). The IC50 is very similar for these three cell lines despite the disparate origins of the tumors within the oral cavity. Induction of apoptosis TPL has been reported to have an anti-tumor effect by apoptosis induction in various solid tumors. Thus, we examined here if it resulted in oral cancer cell death via apoptosis. KB, SCC25 and OECM1 cancer cells were treated with TPL (20–80 nM) for 24 h followed by annexin V/PI staining to examine the proportion of apoptotic cells. It revealed that TPL caused cell apoptosis in a dosedependent manner (Fig. 2A–D), however, there was no apparent effect of necrosis (V /PI+ population) on these cells. Therefore, we suggest annexin V+/PI+ population might be apoptotic cells under secondary necrosis and revealed dose-dependent manner also.

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Enhancement of caspase activities The apoptosis is initiated by a caspase cascade. We therefore addressed whether TPL induced apoptosis by caspase signalling. KB, SCC25 and OEC-M1 cancer cells were treated with 40 nM TPL for 48 h and cell lysates were examined for caspase-8, 9 and 3 activities. Caspase-8, which is activated downstream of the death receptor signaling or via death receptor-independent pathway, was activated through 24–48 h in these cancer cells compared to control groups. (i.e. KB: 1.46 fold and 3.79 fold at 24 h and 48 h, respectively compared with control only; SCC25: 1.79 fold and 5.49 fold; OEC-M1: 1.42 fold and 6.32 fold) (Fig. 3A). Caspase-9, which is activated through the mitochondrial signaling, was determined to be activated in these cells through time (ie. KB: 1.94 fold and 6.3 fold; SCC25: 1.94 fold and 11.21 fold; OEC-M1: 2.25 fold and 7.9 fold) (Fig. 3B). Caspase-3, convergent with a variety of death signals, plays a key role in the induction of apoptosis. TPL also activated caspase-3 in a time-dependent manner. From 12 to 48 h upon TPL exposure, caspase-3 was activated from 4.1 to 119.26 fold in KB cells at indicated time point respectively; 4.08 to 70.18 fold in SCC25 cells; 7.46–90.49 fold in OEC-M1 cells (Fig. 3C). These results revealed that TPL induced caspase cascade and resulted in apoptosis in these cells.

Figure 3 TPL induces caspase activities. KB, SCC25 and OEC-M1 were treated with 40 nM TPL; caspase-8 (A), caspase-9 (B), caspase-3 (C) activity were detect at indicated time points (0–48 h). Activity fold indicates the relative value compared with control groups (*, p < 0.05).

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Week after tumor cells inoculation Figure 4 Tumor growth inhibition in vivo. (A) Daily treatment with TPL (0.15 mg/kg) after tumor xenograft transplantation reduced tumor size compared with vehicle. (B) The average of tumor weight was compared between TPL treated or not tumor-bearing mice on day 15 (KB), day 42(SCC25) and day 84(OEC-M1) (n = 6; *, p < 0.01). Tumor volume of KB (C), SCC25 (D) and OEC-M1 (E) xenograft treated with TPL in vivo or not was measured at indicated time (n = 6).

Anti-tumor effect in vivo Following the investigation of apoptosis induction in oral cancer and KB cells in vitro, the anti-tumor effect of TPL was evaluated. We establish the xenograft tumor-bearing model by separately transplanting KB, SCC25 or OEC-M1 cancer cells into NOD/SCID mice. TPL was first injected on day 3, before the tumor was palpated, in each group of mice, and continuously administrated until day 15, 42, and 84 in KB-, SCC25-, and OEC-M1-bearing mice, respectively, the tumor size was significantly reduced (Fig. 4A). KB xenografts reduced in weight by 87.35 ± 5.78%, SCC25 by 56.42 ± 7.75%, and OEC-M1 by 87.47 ± 5.12% upon TPL treatment (Fig. 4B). The in vivo growth rates of these cells are different, since KB, SCC25, and OEC-M1 tumors were palpable in each group of mice on days 6, 24, and 42, respectively. However, by dynamically measuring tumor volume, TPL clearly showed an inhibitory effect on oral cancers (Fig. 4C–E). These results revealed that TPL is effective against oral cancer. To elucidate whether TPL exerted an antitumor effect in vivo via apoptosis, we examined the tumor by TUNEL analysis. The tumors from TPL treated mice exhibited a markedly higher count of apoptotic cells (TUNEL stain positive) compared with the control tumors (Fig. 5A). The apoptotic index (AI) was significantly increased by treatment with TPL compared with the control group (Fig. 5B). The incidence of apoptosis in the tumor corresponded to the effect of tumor growth inhibition, suggesting that TPL resulted in tumor inhibition by augmentation of apoptosis in the tumor. Discussion TPL, an ancient Chinese herb, has been determined to have significant cytotoxic effect on different types of tumors;13,19,20 how-

ever, its effect on oral cancer has not yet been addressed. In this study, we demonstrate that TPL inhibits oral cancer and KB cell growth by inducing the cell death at only nanomolar level of concentration (Fig. 1); in contrast, the same dose of TPL (less than 20 nM) does not affect normal cell proliferation, such as human lung fibroblast MRC-5 and Swiss mouse embryonic fibroblast 3T3 (data not shown). It reveals that TPL resulted in apoptosis (annexin V+/PI ) but not necrosis (annexin V /PI+) of treated cells in a dose-dependent manner (Fig. 2A–D). One population, stained as annexin V+/PI+, might be undergoing secondary necrosis from the population of early apoptotic (annexin V+/PI ) cells.21 These morphological features of apoptotic versus necrotic cell death can be distinguished under microscopy.21 It suggests that if early apoptotic cells are not ingested by phagocytes in time, secondary necrosis would proceed then.22 Therefore, pooling these secondary necrosis cells with annexin V+/PI cells, the extent of apoptosis resulted by TPL is apparently much greater. The apoptosis induction in oral cancer and KB cells is further confirmed by TPL-mediated caspase activation. Both caspase-8 and caspase-9 are activated in response to TPL in KB, SCC25 and OEC-M1 cells (Fig. 3A and B), suggesting extrinsic and intrinsic apoptotic signaling are both activated by TPL. However, caspase8 is investigated to be involved in death receptor-independent pathway,23,24 and is driven by caspase-3 induced mitochondrial amplification loops. In fact, we observed reduction of caspase-8 activity accompanied with caspase-3 arrest using CPP32 Inhibitor (Ac-DEVD-CHO) and TPL compared with TPL treated only (data not shown). It is also revealed in leukemia cells, TPL induces caspase-dependent cell death only mediated via the mitochondrial (caspase-9) pathway.14 TPL also activates caspase-3 activity in these oral cancer cells whether they carry a p53 mutation or not

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Figure 5 Induction of apoptosis in xenografted tumor cells. (A) KB, SCC25 and OEC-M1 xenograft treated with TPL daily or not was examined on day 15 (KB), day 42 (SCC25), and day 84 (OEC-M1) for apoptosis induction by TUNEL assay under 100 magnifications (scale bars, 100 lm). (B) The apoptotic index (AI) was calculated as the percentage of positive staining cells: AI = number of apoptotic cells/total number of nucleated cells. The apoptotic index indicates the relative value compared with control groups (*, p < 0.05).

(KB: p53 WT;25,26 SCC25 and OEC-M1 p53 mutant27,28) (Fig. 3C) This is in accordance with the previous observation that TPL induces apoptosis in both p53-wild type HeLa (cervical cancer) cells29 and p53-deficient HL-60 (leukemic) cells.30 Therefore, we suggest that TPL induces apoptosis of oral cancer cells through a p53-independent pathway. Although TPL is examined as non-cytotoxic at less than 20 nM on normal cells in vitro, significant cytopathic effect is revealed upon daily administration into NOD/SCID with higher dose than 0.75 mg/kg. Weakness, molt, severe body weight loss and death were obvious in our experiment. However, the tolerated dose less than 0.6 mg/kg of TPL injection does not appear to adversely affect the mice. No apparent signs of sickness and no difference in the body weight loss between groups treated with TPL and the vehicle controls were evident after 2 weeks. Daily treatment with TPL is required because it has a short half-life.15 In the xenograft tumor-bearing mouse model, TPL is revealed to have significant anti-cancer effect (Fig. 4). The therapeutic effect has been confirmed to occur, at least in part, through apoptosis induction, as determined by TUNEL staining of tumor sections (Fig. 5). These oral cancer cell line-bearing mouse models are shown to have degrees of susceptibility to TPL treatment; however, the therapeutic effect is exclusively correlated with caspase-3 activity induced ability by TPL in these cancer cells (Fig. 3). It implies that apoptosis induction is a critical function of TPL to exert its anti-cancer effect on oral cancer and KB cells.

Both chemotherapy and radiotherapy play significant and crucial roles in clinical anti-tumor therapy to prolong patient survival in advanced cancer.31,32 Since the lesions of oral cancer are easy to access, the combination of chemotherapy and radiation may improve local tumor control in cases of non-resectable tumors or tumors that are non-responsive to chemotherapy or radiation alone.32 TPL is capable of acting synergistically with conventional chemotherapeutic drugs doxorubicin on fibrosarcoma cell lines and 5-fluorouracil on colon carcinoma.13,15,33 Another interesting report showed that combining the use of TPL and ionizing radiation exhibits enhanced antitumor effect.34 It is now proven that TPL also has anti-cancer effect on oral cancer, and further efforts are worthwhile to combine this potentate herb with other adjuvant therapy in advanced oral cancer. In conclusion, our study is the first to determine that TPL exerts its anti-cancer effect on oral cancer via caspase-dependent apoptosis. These results may lead to the usage of TPL on chemotherapy and chemoprevention in oral cancer in the future. Conflict of Interest Statement None declared. Acknowledgement This study was supported by research grant from Tri-Service General Hospital, Republic of China and Grant No. TSGH-C98-27.

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