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Comparisons of norcantharidin cytotoxic effects on oral cancer cells and normal buccal keratinocytes
Oral Oncology 39 (2003) 19–26 www.elsevier.com/locate/oraloncology
Comparisons of norcantharidin cytotoxic effects on oral cancer cells and normal buccal keratinocytes S.H. Koka,b,1, C.Y. Honga,b,1, M.Y.P. Kuoa,b, C.H.K. Leea, J.J. Leeb, I.U. Loua, M.S. Leea, M. Hsiaoc, S.K. Lina,b,* a
School of Dentistry, National Taiwan University Medical Center, 1 Chang-Teh Street, Taipei, Taiwan 10016, ROC b Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan, ROC c Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaoshiung, Taiwan, ROC Received 25 October 2001; accepted 28 November 2001
Abstract Norcantharidin (NCTD) is the demethylated analogue of cantharidin. In this study, multi-parameter assessments of morphological alterations, clonogenic efficiency, cell growth curves, DNA synthesis, and DNA strand break were employed to determine and compare the cytotoxic effects of NCTD on oral cancer KB cell line and normal buccal keratinocytes. The results showed NCTD induced significant cytotoxicity in KB cells after 24 h of exposure. Normal buccal keratinocytes were more resistant to NCTD induced cytotoxicity. The IC50 of 24 h NCTD treatment for KB and keratinocytes were 15.06 and 216.29 mg/ml, respectively with a keratinocyte/KB selective index of 14.36. Anoikis and membrane blebbing, morphological characterization of apoptosis, were observed in about 90% of KB cells after exposure to 100 mg/ml of NCTD for 24 h compared to about 30% in keratinocytes. In addition, inhibition of colony formation was noted in KB cells even when exposed to low concentration of drug (5 mg/ml) for a short period of time (6 h). NCTD inhibited subsequent cell proliferation in KB but growth of normal keratinocytes was retarded only temporarily. NCTD inhibited DNA synthesis in both KB and normal keratinocytes. However, keratinocytes were more sensitive to DNA synthesis inhibition by low dose of NCTD. Significant DNA strand break was noted in KB cells only after cell viability was reduced to less than 60% of the control. In comparison, normal keratinocytes were resistant to NCTD induced DNA strand break. These results indicated KB cells were more sensitive to NCTD induced cytotoxicity compared to normal keratinocytes. NCTD may be of value in treating oral cancers. The underlying mechanisms of the differential actions of NCTD on these two cell types are worthy of further investigations. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Norcantharidin; Oral cancer; Buccal keratinocytes; Cytotoxicity
1. Introduction Oral cancer is the sixth most common cancer globally [1]. In the Far East and South Asian countries the incidence is much higher where oral cancers constitute up to 25% of all malignancies [1]. Squamous cell carcinoma is observed to be the most common malignant lesion in the oral cavity where the most important risk factors are linked to tobacco and alcohol [2]. The chewing of areca nut (Areca catechu) preparations has also been thought to associate with the high incidence of oral cancer observed in India and many Southeast Asian countries * Corresponding author. Tel.: +886-2-23123456 ext. 7337; fax: +886-2-23831346. E-mail address: [email protected] (S.K. Lin). 1 The first two authors contributed equally to this paper.
[3]. Although early stage disease is highly curable, more than 50% of oral cancer patients present with advanced disease [2]. In spite of the improvement in the diagnostic and treatment technology, fewer than 30% of patients with advanced diseases are cured [2]. Furthermore, oral cancer has a high rate of second primary tumors occurring in patients previously considered cured. It is obvious that a new therapeutic approach to this disease is needed. Recent meta-analyses [4,5] showed that concurrent chemoradiotherapy programs have resulted in modest but significant gains in the overall survival of patients with squamous cell carcinoma of the head and neck, especially for patients with oral cavity tumors [6]. However, no specific chemoradiotherapy program has emerged as the definitive acceptable standard of care and there is much room for improvement. Continued investigation of new chemotherapeutic agents is thus needed.
1368-8375/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S1368-8375(01)00129-4
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Nomenclature NCTD norcantharidin
Cantharidin, a 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative, is a natural toxin extracted from blister beetles and the active ingredient of the purported aphrodisiac Spanish fly [7]. It has been demonstrated to have strong inhibitory effect on protein phosphatases type 2A (PP2A) [8,9]. It inhibits some tumor cell types such as HeLa cells, murine ascites hepatoma, or reticular cell sarcoma but not murine erythroid leukemia cells in vitro, S180 in vivo, and Walker tumor in rats [10]. Clinical trials indicated that cantharidin had effects on patients with primary hepatoma, but the application was limited by its severe toxicity for mucous membranes, mainly in the gastrointestinal tract, ureter and kidney [10]. A series of bioactive analogues have been synthesized in an attempt to increase the utility and to reduce the toxicity of cantharidin. Norcantharidin (NCTD), the demethylated analogue of cantharidin, appeared to cause the least nephrotoxic and inflammatory side effects. NCTD inhibits the proliferation of a variety of human tumor cell lines [10–12]. Yang et al. [13] have also demonstrated that NCTD inhibited the growth of transplanted human hepatocellular carcinoma and prolonged host survival. It is noteworthy that NCTD can stimulate the bone marrow and increase the peripheral WBC significantly [14]. This function of NCTD make this compound valuable in combination chemotherapy since most of the chemotherapeutic agents in use today have the side effect of bone marrow suppression. Although NCTD was shown to be effective in inhibiting tumor cell proliferation and was used to treat human cancers in China since 1984 [10], there were, however, very few reports describing the cytotoxic effects of NCTD on tumor cells. In this study, we investigated the cytotoxic effects of NCTD on oral cancer cells and normal keratinocytes. Human oral cancer cell line KB and cultured buccal keratinocytes were used as target cells. The toxicity of NCTD was assessed by acute cell death and concomitant morphological alterations, clonogenic efficiency, cell growth curve, DNA synthesis and DNA strand break analyses.
2. Materials and methods 2.1. Cell lines and cell cultures KB cells (ATCC CCL-17; American Type Culture Collection, Rockville, MD, USA) were cultivated in 5% CO2 at 37 C in high glucose DMEM (Gibco/BRL,
Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco/BRL) and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin; Gibco/BRL). Healthy buccal mucosa was obtained from four volunteer dental students with proper informed consent. The collected tissue was incubated in 0.25% trypsin/Tris–saline for 2 h to facilitate the removal of connective tissue [15]. The epithelial tissues were cut into small pieces and cultured by an explant technique described previously [16]. The explants were cultured at 37 C in 5% CO2 on collagen(80 mg/ml; calf skin type I collagen, Sigma Chemical Company, St Louis, MO, USA)-coated plates with keratinocyte growth medium (KGM-SFM, Gibco/ BRL) containing 5 ng/ml of EGF (epidermal growth factor, Gibco/BRL) and 50 mg/ml of pituitary gland extract (Gibco/BRL). The buccal keratinocytes from passage 1 to 3 were used for this study. 2.2. Preparation of norcantharidin NCTD is the demethylated analogue of cantharidin, a 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative. NCTD is synthesized from furan and maleic anhydride via the Diels-Alder reaction. It is purchased from Beijing Fourth Pharmaceutical Works, China. Serial dilutions of NCTD in culture medium were prepared. For MTT assay, 10-fold dilutions from 1000 to 0.1 mg/ ml were used. The other assays were performed with 0, 5, 10, 25 and 50 mg/ml solutions. 2.3. MTT assay KB cells and normal keratinocytes in the appropriate medium were seeded (5104 cells/well) into 24-well culture plates and incubated for 24 h. The medium (1 ml) was then substituted by one containing various concentrations of NCTD (0.1–1000 mg/ml) and further incubated for 6 or 24 h. At the end of incubation the medium was changed and the morphological changes of the cultured cells were observed and photographed by a Nikon phase contrast microscope. The relative cell number was then determined with the MTT assay as described by Mosmann [17]. Briefly, 0.5 mg of the tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl-tetrazolium bromide; Sigma, St Louis, MO, USA) was added to each well containing fresh medium and incubated for 2 h. The insoluble formazan produced was lysed with 1 ml DMSO and the optical density (OD) was read against a reagent blank with a Dias Microplate Well Reader (Dynatech Medical Products Ltd, UK) at a wave length of 540 nm. The OD540 value of the experimental groups was divided by the OD540 value of untreated controls and presented as a percentage. Three separate experiments were performed. The concentrations of drug giving 50% growth inhibition (IC50) were obtained from the dose-response curves
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generated by a non-linear fitting procedure with the statistical package (STATISTICA version 5.1, StatSoft, Tulsa, OK, USA). 2.4. Clonogenic assay Because of the low clonogenic capacity of the normal keratinocytes, clonogenic assay was performed only for KB cells. Cells were seeded into 60-mm plastic dishes in 5 ml of complete media at a density of 250 cells/dish. After 24 h, appropriate amount of NCTD (0, 5, 10, 25 or 50 mg/ml) were added to the dishes, and the cells were exposed for 6 and 24 h. Thereafter, the cells were washed, fresh drug-free medium was added and incubated for 10 days under growth conditions. Finally, cells were stained in 10% Giemsa solution and the numbers of colonies counted. Colonies of at least 50 cells were scored [18]. Each experiment was performed three times using triplicate cultures.
at an initial density of 5104 cells/well into 24-well culture plates and incubated for 24 h. Cells were radiolabeled with 0.2 mCi/ml/well [methyl-3H]-thymidine (Amersham, UK) for 24 h. The radioactive medium was discarded and the cells were treated with different doses of NCTD (0, 5, 10, 25 or 50 mg/ml) in fresh medium for 6 and 24 h. The cells were washed twice with PBS and lysed with 400 ml of lysis buffer (2% SDS, 10 mM EDTA, 10 mM Tris, 0.05 M NaOH, pH 12.4), followed by gently addition of 400 ml of 120 mM KCl into the wells, and the culture plates were incubated at 65 C for 10 min. The cell extracts were transferred to Eppendorf tubes and kept on ice for 5 min. Centrifugation at 3500 rpm for 10 min at 10 C to separate the DNA precipitants and supernatants was done, followed by measurement of radioactivity of the supernatants and pellets by a Beckman liquid scintillation counter. The pellet count was divided by the total counts and expressed as a percentage of DNA precipitated. Triplicate experiments were performed.
2.5. In vitro growth curves 2.8. Statistical analysis KB cells and normal keratinocytes were diluted in appropriate medium, seeded in 6-well plates (105 cells/ well) and cultured for 24 h. The medium was substituted by one containing various concentrations of NCTD (0, 5, 10, 25 or 50 mg/ml) and incubated for 6 or 24 h. After PBS wash, fresh medium was added and the cells were further cultured for 1, 2, 3, 4 and 5 days. At the end of incubation, the cells were washed with PBS, harvested by trypsinization and counted in a hemocytometer. Three separate experiments were performed for each concentration/exposure time combination.
Three separate experiments were performed for each condition in the study. Means and standard deviations were calculated. The results were analyzed by Student’s t-test. A P value of < 0.05 was considered statistically significant.
3. Results 3.1. KB oral cancer cells showed 14 fold more sensitivity to NCTD killing compared to normal keratinocytes
2.6. 3H-thymidine incorporation assay The cells were plated at an initial density of 5104 cells/well into 24-well culture plates and incubated for 24 h. After treatment with different doses of NCTD (0, 5, 10, 25 or 50 mg/ml) for 6 or 24 h, the fresh medium supplemented with 1 mCi/ml/well [methyl-3H]-thymidine (Amersham, UK) was added for a further 24 h. On termination, medium was removed, the cells were washed with PBS, and DNA was then precipitated with ice-cold 10% trichloroacetic acid. The cell layers were lysed with 300 ml of 2 M perchloric acid (PCA) at 60 C for 1 h. The radioactivity of PCA extract was measured by a Beckman liquid scintillation counter (Beckman Instruments, USA). Triplicate experiments were performed. 2.7. DNA strand breakage assay DNA strand breakage was measured by DNA precipitation assay as described by Olive [19] with modifications by Martins et al. [20]. Briefly, cells were plated
KB cells and normal keratinocytes were exposed to various concentrations of NCTD (0.1 to 1000 mg/ml) for 6 and 24 h. Cell survival was measured by MTT assay and the IC50 values were determined. Table 1 summarized the IC50 values of NCTD on KB cells and normal keratinocytes after different treatment periods. KB cells were found to be more sensitive to NCTD compared to normal keratinocytes. The IC50 values for KB cells after 6 and 24 h treatment with NCTD were 209.97 and 15.06 mg/ml respectively, whereas the values for normal keratinocytes were 3865.69 and 216.29 mg/ml (Table 1). The selective indices (SI; the ratios of IC50 values of normal Table 1 IC50 valuesa (mg/ml) of NCTD on KB cells and normal keratinocytes Treatment period
KB cells
Normal keratinocytes
SIb
6h 24 h
209.97 15.06
3865.69 216.29
18.41 14.36
a b
Data represent the mean values of three separate experiments. SI, selective index, IC50 of normal keratinocytes/IC50 of KB cells.
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keratinocytes to that of KB cells) of NCTD treatment for 6 and 24 h were 18.41 and 14.36, respectively. This means KB cells were more than 14 fold more susceptible to NCTD induced cytotoxicity compared to normal keratinocytes. 3.2. NCTD induced significant anoikis and membrane blebbing in KB cells Phase-contrast microscope observations showed the morphological alterations of the cells after exposure to NCTD (Fig. 1). Untreated normal keratinocytes (Fig. 1A) and KB cells (Fig. 1D) were polygonal or
ovoid in appearances with well-defined intercellular spaces. The KB cells were more densely packed on the culture plates. Following exposure to 10 mg/ml of NCTD for 24 h, a significant number of KB cells were roundedup (Fig. 1E). In contrast, only very few keratinocytes showed round cell morphology 24 h after adding 10 mg/ ml of NCTD (Fig. 1B). Treatment of KB cells with 100 mg/ml of NCTD for 24 h resulted in about 90% of cells undergoing anoikis and membrane blebbing (Fig. 1F). In comparison, the keratinocytes showed round cell morphology in only about 30% of cells after 24 h exposure to 100 mg/ml of NCTD (Fig. 1C). Most of the keratinocytes were found attached to the culture plate.
Fig. 1. Morphological alterations in buccal keratinocytes and KB cells following exposure to NCTD for 24 h. (A) Untreated keratinocytes showing polygonal appearances, intercellular spaces are readily demonstrated (arrowheads). (B) Exposure of normal keratinocytes to 10 mg/ml of NCTD. Some cells are rounded (arrowheads) in appearance. (C) Exposure of normal keratinocytes to 100 mg/ml of NCTD. More cells are rounded-up (arrowheads) and some cells have cell membrane protrusions (arrow), however, most cells remain attached to the plate. (D) Untreated KB cells, polygonal and ovoid with well-defined intercellular spaces (arrowheads). (E) Exposure of KB cells to 10 mg/ml of NCTD. A significant number of cells are round (arrowheads). (F) Exposure of KB cells to 100 mg/ml of NCTD. Most cells are round (arrowheads) and membrane blebbing is readily noted (arrows; All200).
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3.3. NCTD reduced colony forming ability of KB cells in a dose and time-dependent manner Clonogenic survival was studied in KB cells treated with different doses of NCTD (0, 5, 10, 25 or 50 mg/ml). NCTD demonstrated a clear negative effect on colonyforming ability compared to controls (Fig. 2). NCTD treatment resulted in a dose and exposure time-dependent decline in colony counts. Significant clonogenic inhibition was noted even when the cells were exposed to a low concentration (5 mg/ml) of drug for a short period of time (6 h). 3.4. Significant growth retardation induced by NCTD in KB cells but not in keratinocytes KB cells and normal keratinocytes were treated with various concentrations of NCTD (0, 5, 10, 25 or 50 mg/ ml) for 6 and 24 h and the effect on subsequent growth of the survived cells was observed. Fig. 3 shows marked growth retardation of KB cells following exposure to NCTD. For 6 h exposure, lower concentrations (5 and 10 mg/ml) of NCTD caused significant inhibition of cell growth in the early 3 or 4 days but a delayed recovery was still noted (Fig. 3A). With higher concentrations, NCTD inhibited cell growth completely (50 mg/ml) or only a weak recovery was noted (25 mg/ml) in the later days of the experiment. When treated for a longer time (24 h), NCTD inhibited the growth of KB cells to a large extent (Fig. 3B). A delayed recovery was noted only for the lowest concentration (Fig. 3B, 5 mg/ml) and higher concentrations of NCTD (Fig. 3B, 25 mg/ml, and 50 mg/ml) resulted in almost complete inhibition of cell growth throughout the assay period (day 1–day 5). NCTD treatment for 6 and 24 h reduced the numbers of
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normal keratinocytes to a certain extent. However, the subsequent growth of the survived keratinocytes was largely not inhibited (Fig. 4). Only transient growth inhibition was noted in the buccal keratinocytes treated with up to 50 mg/ml of NCTD for 6 (Fig. 4A) or 24 h (Fig. 4B). Cell growth of the NCTD-treated keratinocytes caught up with that of the untreated controls on the third or fourth day after drug exposure. 3.5. Buccal keratinocytes were more susceptible to NCTD induced DNA synthesis inhibition The effect of NCTD on [methyl-3H]-thymidine incorporation in KB cells and normal keratinocytes were studied. NCTD inhibited de novo DNA synthesis in a dose and exposure time-dependent manner (Fig. 5). At lower concentrations (5 and 10 mg/ml), 6 h NCTD treatment inhibited DNA synthesis to a similar extent in KB cells and normal keratinocytes but the inhibition was significantly more marked in normal keratinocytes after a 24 h-exposure (Fig. 5). At higher concentrations NCTD further reduced the incorporation of 3H-thymidine and DNA synthesis was almost completely abolished in both normal keratinocytes and KB cells by 6 and 24 h exposure to 50 mg/ml of NCTD (Fig. 5). 3.6. Higher concentrations of NCTD induced significant DNA strand break in KB cells but not in keratinocytes DNA strand breaks were determined in KB cells and normal keratinocytes by DNA precipitation assay after treatment with various concentrations of NCTD. The amount of DNA released from the nucleoprotein (supernatant) was compared to that still attached to the protein (precipitate). The proportion of DNA remaining in the supernatant was a measure of DNA damage in the cells. As shown in Fig. 6, significant DNA damage was noted only in KB cells after exposing the cells to higher concentrations (510 mg/ml) of NCTD for a long period (24 h). A 6 h-exposure to NCTD resulted in no significant DNA damage in KB cells regardless of the concentrations used. NCTD caused no significant DNA damage in normal keratinocytes with the doses and exposure time used in the experiment.
4. Discussion
Fig. 2. Clonogenic assay of KB cells after NCTD treatment. The cells were treated with various concentrations of NCTD for 6 and 24 hr. The numbers of colonies (at least 50 cells) were counted after 10 days of growth. Results are expressed as percentages of colony number relative to the untreated control. Each point represents the mean of triplicate experiments. Bars, S.D. *P <0.05 vs. control (0 mg/ml).
The role of chemotherapy in treating head and neck cancer has been expanding. In recent years, concurrent chemoradiotherapy programs have resulted in clinically and statistically significant gains in the management of squamous cell carcinoma of the head and neck [4,5]. However, each program has its risks and benefits and myelosuppression remains the major limiting toxicity for most of the regimens in use today [21]. Conse-
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Fig. 3. Growth curves of KB cells after NCTD treatment. The cells were treated with various concentrations of NCTD for 6 (A) and 24 hr (B). The cells were washed and fresh medium was added. The numbers of cells were counted in a hemocytometer 0, 1, 2, 3, 4 and 5 days after drug removal. Each point represents the mean of triplicate experiments. Bars, S.D. *P<0.05 vs. control (0 mg/ml).
Fig. 4. Growth curves of normal buccal keratinocytes after NCTD treatment. The cells were treated with various concentrations of NCTD for 6 (A) and 24 hr (B). The cells were washed and fresh medium was added. The numbers of cells were counted in a hemocytometer 0, 1, 2, 3, 4 and 5 days after drug removal. Each point represents the mean of triplicate experiments. Bars, S.D. *P <0.05 vs. control (0 mg/ml).
quently, it is necessary to continue searching for cytotoxic agents to tumor cells with improved or altered efficacy profile and minimum toxicity to hematopoietic system and normal tissues. NCTD has been shown to be effective in inhibiting tumor cell proliferation in addition to its ability to stimulate the bone marrow activity [10,14]. Although it was used to treat cancers in China since 1984 [10], its cytotoxic effects on oral cancer cells has not been reported. In the present study, we showed that NCTD can cause acute toxicity to oral cancer cells. Normal oral keratinocytes are relatively resistant to NCTD treatment (SI for 6 and 24 h treatment were 18.41 and 14.36, respectively). The IC50 values of NCTD on KB cells were comparable to those determined for other tumor cell lines. McCluskey
et al. [12] recently reported that the IC50 values of various tumor cell lines derived from ovary, colon and osteosarcoma after 72 h exposure to NCTD were in the range of 20 to 50 mM. Our results showed the IC50 of KB cells after NCTD treatment for 24 h was 15.06 mg/ ml (89.6 mM). The cytotoxicity of NCTD was further demonstrated by its negative effect on the colony-forming ability of KB cells. Clonogenic inhibition was obvious even after a short exposure (6 h) to 5 mg/ml of NCTD. Cell shrinkage and membrane blebbing were observed after NCTD treatment and such alterations are compatible with the apoptotic morphology [22]. These results are comparable to those in our previous report showing NCTD induced similar morphologic changes and apoptosis in glioblastoma cells [11].
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Fig. 5. Inhibition of DNA synthesis in normal keratinocytes and KB cells by NCTD. The cells were treated with various concentrations of NCTD for 6 and 24 hr and the incorporation of 3H-thymidine was assayed. Results are expressed as percentages of 3H-thymidine incorporation relative to the untreated control. Each point represents the mean of triplicate experiments. Bars, S.D. *P<0.05 vs. control (0 mg/ ml).
The kinetics of cell growth after NCTD exposure revealed interesting differences between KB and normal keratinocytes. Normal keratinocytes could recover from the drug treatment and their growth caught up with that of the untreated cells even after exposure to 50 mg/ml of NCTD for 24 h. In contrast, significant growth retardation was noted after KB oral cancer cells treated with as low as 10 mg/ml of NCTD for 24 h. The exposure of KB cells to 50 mg/ml of NCTD for 6 or 24 h resulted in complete inhibition of subsequent cell growth (Fig. 2). These after-effects may be attributed to irreversible changes in cellular functions induced by NCTD that lead to delayed cell death. The exact mechanism of the selective cytotoxicity of NCTD on normal buccal keratinocytes and oral cancer cells needs further investigation. NCTD inhibited DNA synthesis in a dose and exposure time-dependent manner. It is interesting to find out that NCTD blocked DNA synthesis to a larger extent in normal keratinocytes than that in KB cells. The concentration resulted in 50% inhibition of 3H-thymidine incorporation in normal keratinocytes (5.70 mg/ml) was lower than the IC50 for acute cell death (216.29 mg/ml) after 24 h NCTD exposure. In KB cells, the 50% inhibitory concentration of 3H-thymidine incorporation (19.73 mg/ml) was similar to the IC50 for acute cell death (15.06 mg/ml) when treated with NCTD for 24 h. The results indicated that NCTD caused transient growth arrest in normal keratinocytes since cell growth could catch up with the untreated control several days after drug removal. The signaling pathways underlying the selection between cell death and growth arrest should be addressed by future investigations on NCTD cytotoxicity. Many of the chemotherapeutic agents in use today are genotoxic. In our study, NCTD induced DNA damage only in KB cells at higher concentrations (510 mg/ml)
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Fig. 6. DNA strand breaks in normal keratinocytes and KB cells induced by NCTD. The cells were treated with various concentrations of NCTD for 6 and 24 hr and DNA strand breaks were measured by DNA precipitation assay. Radioactivity of the supernatants (which contained DNA strand breaks) and the precipitates (which contained bulk DNA) was measured separately. The radioactivity of the precipitates was expressed as percentages of total radioactivity. Each point represents the mean of triplicate experiments. Bars, S.D. *P <0.05 vs. control (0 mg/ml) 2–4.
and longer exposure (24 h). DNA strand break was seen only after cell viability was reduced to less than 60% of the control, indicating that DNA strand break was the consequence of extragenomic damage and viability loss [23]. In conclusion, our results showed that NCTD is more effective in inducing cytotoxicity in oral cancer cells compared to normal buccal keratinocytes as demonstrated by acute cell death and cell growth inhibition assays after drug treatment. Oral cancer cells are more readily killed by NCTD and their subsequent growth is inhibited even after drug removal. On the other hand, NCTD can cause temporary growth arrest in normal keratinocytes. The differential toxicity of NCTD on oral cancer cells and buccal keratinocytes may be of value to its clinical application.
Acknowledgements This work was supported in part by grants 89-2314-B002-366 (Lin) from National Science Council and DOH89-TD-1134, DOH90-TD-1041 (Hong) from National Health Research Institute, Taiwan, ROC. References [1] Notani PN. Epidemiology and prevention of head and neck cancer: a global view. In: Saranath D, editor. Contemporary Issues in Oral Cancer. New Delhi: Oxford University Press, 2000. p. 1–29.
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Comparisons of norcantharidin cytotoxic effects on oral cancer cells and normal buccal keratinocytes S.H. Koka,b,1, C.Y. Honga,b,1, M.Y.P. Kuoa,b, C.H.K. Leea, J.J. Leeb, I.U. Loua, M.S. Leea, M. Hsiaoc, S.K. Lina,b,* a
School of Dentistry, National Taiwan University Medical Center, 1 Chang-Teh Street, Taipei, Taiwan 10016, ROC b Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan, ROC c Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaoshiung, Taiwan, ROC Received 25 October 2001; accepted 28 November 2001
Abstract Norcantharidin (NCTD) is the demethylated analogue of cantharidin. In this study, multi-parameter assessments of morphological alterations, clonogenic efficiency, cell growth curves, DNA synthesis, and DNA strand break were employed to determine and compare the cytotoxic effects of NCTD on oral cancer KB cell line and normal buccal keratinocytes. The results showed NCTD induced significant cytotoxicity in KB cells after 24 h of exposure. Normal buccal keratinocytes were more resistant to NCTD induced cytotoxicity. The IC50 of 24 h NCTD treatment for KB and keratinocytes were 15.06 and 216.29 mg/ml, respectively with a keratinocyte/KB selective index of 14.36. Anoikis and membrane blebbing, morphological characterization of apoptosis, were observed in about 90% of KB cells after exposure to 100 mg/ml of NCTD for 24 h compared to about 30% in keratinocytes. In addition, inhibition of colony formation was noted in KB cells even when exposed to low concentration of drug (5 mg/ml) for a short period of time (6 h). NCTD inhibited subsequent cell proliferation in KB but growth of normal keratinocytes was retarded only temporarily. NCTD inhibited DNA synthesis in both KB and normal keratinocytes. However, keratinocytes were more sensitive to DNA synthesis inhibition by low dose of NCTD. Significant DNA strand break was noted in KB cells only after cell viability was reduced to less than 60% of the control. In comparison, normal keratinocytes were resistant to NCTD induced DNA strand break. These results indicated KB cells were more sensitive to NCTD induced cytotoxicity compared to normal keratinocytes. NCTD may be of value in treating oral cancers. The underlying mechanisms of the differential actions of NCTD on these two cell types are worthy of further investigations. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Norcantharidin; Oral cancer; Buccal keratinocytes; Cytotoxicity
1. Introduction Oral cancer is the sixth most common cancer globally [1]. In the Far East and South Asian countries the incidence is much higher where oral cancers constitute up to 25% of all malignancies [1]. Squamous cell carcinoma is observed to be the most common malignant lesion in the oral cavity where the most important risk factors are linked to tobacco and alcohol [2]. The chewing of areca nut (Areca catechu) preparations has also been thought to associate with the high incidence of oral cancer observed in India and many Southeast Asian countries * Corresponding author. Tel.: +886-2-23123456 ext. 7337; fax: +886-2-23831346. E-mail address: [email protected] (S.K. Lin). 1 The first two authors contributed equally to this paper.
[3]. Although early stage disease is highly curable, more than 50% of oral cancer patients present with advanced disease [2]. In spite of the improvement in the diagnostic and treatment technology, fewer than 30% of patients with advanced diseases are cured [2]. Furthermore, oral cancer has a high rate of second primary tumors occurring in patients previously considered cured. It is obvious that a new therapeutic approach to this disease is needed. Recent meta-analyses [4,5] showed that concurrent chemoradiotherapy programs have resulted in modest but significant gains in the overall survival of patients with squamous cell carcinoma of the head and neck, especially for patients with oral cavity tumors [6]. However, no specific chemoradiotherapy program has emerged as the definitive acceptable standard of care and there is much room for improvement. Continued investigation of new chemotherapeutic agents is thus needed.
1368-8375/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S1368-8375(01)00129-4
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Nomenclature NCTD norcantharidin
Cantharidin, a 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative, is a natural toxin extracted from blister beetles and the active ingredient of the purported aphrodisiac Spanish fly [7]. It has been demonstrated to have strong inhibitory effect on protein phosphatases type 2A (PP2A) [8,9]. It inhibits some tumor cell types such as HeLa cells, murine ascites hepatoma, or reticular cell sarcoma but not murine erythroid leukemia cells in vitro, S180 in vivo, and Walker tumor in rats [10]. Clinical trials indicated that cantharidin had effects on patients with primary hepatoma, but the application was limited by its severe toxicity for mucous membranes, mainly in the gastrointestinal tract, ureter and kidney [10]. A series of bioactive analogues have been synthesized in an attempt to increase the utility and to reduce the toxicity of cantharidin. Norcantharidin (NCTD), the demethylated analogue of cantharidin, appeared to cause the least nephrotoxic and inflammatory side effects. NCTD inhibits the proliferation of a variety of human tumor cell lines [10–12]. Yang et al. [13] have also demonstrated that NCTD inhibited the growth of transplanted human hepatocellular carcinoma and prolonged host survival. It is noteworthy that NCTD can stimulate the bone marrow and increase the peripheral WBC significantly [14]. This function of NCTD make this compound valuable in combination chemotherapy since most of the chemotherapeutic agents in use today have the side effect of bone marrow suppression. Although NCTD was shown to be effective in inhibiting tumor cell proliferation and was used to treat human cancers in China since 1984 [10], there were, however, very few reports describing the cytotoxic effects of NCTD on tumor cells. In this study, we investigated the cytotoxic effects of NCTD on oral cancer cells and normal keratinocytes. Human oral cancer cell line KB and cultured buccal keratinocytes were used as target cells. The toxicity of NCTD was assessed by acute cell death and concomitant morphological alterations, clonogenic efficiency, cell growth curve, DNA synthesis and DNA strand break analyses.
2. Materials and methods 2.1. Cell lines and cell cultures KB cells (ATCC CCL-17; American Type Culture Collection, Rockville, MD, USA) were cultivated in 5% CO2 at 37 C in high glucose DMEM (Gibco/BRL,
Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco/BRL) and antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin; Gibco/BRL). Healthy buccal mucosa was obtained from four volunteer dental students with proper informed consent. The collected tissue was incubated in 0.25% trypsin/Tris–saline for 2 h to facilitate the removal of connective tissue [15]. The epithelial tissues were cut into small pieces and cultured by an explant technique described previously [16]. The explants were cultured at 37 C in 5% CO2 on collagen(80 mg/ml; calf skin type I collagen, Sigma Chemical Company, St Louis, MO, USA)-coated plates with keratinocyte growth medium (KGM-SFM, Gibco/ BRL) containing 5 ng/ml of EGF (epidermal growth factor, Gibco/BRL) and 50 mg/ml of pituitary gland extract (Gibco/BRL). The buccal keratinocytes from passage 1 to 3 were used for this study. 2.2. Preparation of norcantharidin NCTD is the demethylated analogue of cantharidin, a 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid derivative. NCTD is synthesized from furan and maleic anhydride via the Diels-Alder reaction. It is purchased from Beijing Fourth Pharmaceutical Works, China. Serial dilutions of NCTD in culture medium were prepared. For MTT assay, 10-fold dilutions from 1000 to 0.1 mg/ ml were used. The other assays were performed with 0, 5, 10, 25 and 50 mg/ml solutions. 2.3. MTT assay KB cells and normal keratinocytes in the appropriate medium were seeded (5104 cells/well) into 24-well culture plates and incubated for 24 h. The medium (1 ml) was then substituted by one containing various concentrations of NCTD (0.1–1000 mg/ml) and further incubated for 6 or 24 h. At the end of incubation the medium was changed and the morphological changes of the cultured cells were observed and photographed by a Nikon phase contrast microscope. The relative cell number was then determined with the MTT assay as described by Mosmann [17]. Briefly, 0.5 mg of the tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl-tetrazolium bromide; Sigma, St Louis, MO, USA) was added to each well containing fresh medium and incubated for 2 h. The insoluble formazan produced was lysed with 1 ml DMSO and the optical density (OD) was read against a reagent blank with a Dias Microplate Well Reader (Dynatech Medical Products Ltd, UK) at a wave length of 540 nm. The OD540 value of the experimental groups was divided by the OD540 value of untreated controls and presented as a percentage. Three separate experiments were performed. The concentrations of drug giving 50% growth inhibition (IC50) were obtained from the dose-response curves
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generated by a non-linear fitting procedure with the statistical package (STATISTICA version 5.1, StatSoft, Tulsa, OK, USA). 2.4. Clonogenic assay Because of the low clonogenic capacity of the normal keratinocytes, clonogenic assay was performed only for KB cells. Cells were seeded into 60-mm plastic dishes in 5 ml of complete media at a density of 250 cells/dish. After 24 h, appropriate amount of NCTD (0, 5, 10, 25 or 50 mg/ml) were added to the dishes, and the cells were exposed for 6 and 24 h. Thereafter, the cells were washed, fresh drug-free medium was added and incubated for 10 days under growth conditions. Finally, cells were stained in 10% Giemsa solution and the numbers of colonies counted. Colonies of at least 50 cells were scored [18]. Each experiment was performed three times using triplicate cultures.
at an initial density of 5104 cells/well into 24-well culture plates and incubated for 24 h. Cells were radiolabeled with 0.2 mCi/ml/well [methyl-3H]-thymidine (Amersham, UK) for 24 h. The radioactive medium was discarded and the cells were treated with different doses of NCTD (0, 5, 10, 25 or 50 mg/ml) in fresh medium for 6 and 24 h. The cells were washed twice with PBS and lysed with 400 ml of lysis buffer (2% SDS, 10 mM EDTA, 10 mM Tris, 0.05 M NaOH, pH 12.4), followed by gently addition of 400 ml of 120 mM KCl into the wells, and the culture plates were incubated at 65 C for 10 min. The cell extracts were transferred to Eppendorf tubes and kept on ice for 5 min. Centrifugation at 3500 rpm for 10 min at 10 C to separate the DNA precipitants and supernatants was done, followed by measurement of radioactivity of the supernatants and pellets by a Beckman liquid scintillation counter. The pellet count was divided by the total counts and expressed as a percentage of DNA precipitated. Triplicate experiments were performed.
2.5. In vitro growth curves 2.8. Statistical analysis KB cells and normal keratinocytes were diluted in appropriate medium, seeded in 6-well plates (105 cells/ well) and cultured for 24 h. The medium was substituted by one containing various concentrations of NCTD (0, 5, 10, 25 or 50 mg/ml) and incubated for 6 or 24 h. After PBS wash, fresh medium was added and the cells were further cultured for 1, 2, 3, 4 and 5 days. At the end of incubation, the cells were washed with PBS, harvested by trypsinization and counted in a hemocytometer. Three separate experiments were performed for each concentration/exposure time combination.
Three separate experiments were performed for each condition in the study. Means and standard deviations were calculated. The results were analyzed by Student’s t-test. A P value of < 0.05 was considered statistically significant.
3. Results 3.1. KB oral cancer cells showed 14 fold more sensitivity to NCTD killing compared to normal keratinocytes
2.6. 3H-thymidine incorporation assay The cells were plated at an initial density of 5104 cells/well into 24-well culture plates and incubated for 24 h. After treatment with different doses of NCTD (0, 5, 10, 25 or 50 mg/ml) for 6 or 24 h, the fresh medium supplemented with 1 mCi/ml/well [methyl-3H]-thymidine (Amersham, UK) was added for a further 24 h. On termination, medium was removed, the cells were washed with PBS, and DNA was then precipitated with ice-cold 10% trichloroacetic acid. The cell layers were lysed with 300 ml of 2 M perchloric acid (PCA) at 60 C for 1 h. The radioactivity of PCA extract was measured by a Beckman liquid scintillation counter (Beckman Instruments, USA). Triplicate experiments were performed. 2.7. DNA strand breakage assay DNA strand breakage was measured by DNA precipitation assay as described by Olive [19] with modifications by Martins et al. [20]. Briefly, cells were plated
KB cells and normal keratinocytes were exposed to various concentrations of NCTD (0.1 to 1000 mg/ml) for 6 and 24 h. Cell survival was measured by MTT assay and the IC50 values were determined. Table 1 summarized the IC50 values of NCTD on KB cells and normal keratinocytes after different treatment periods. KB cells were found to be more sensitive to NCTD compared to normal keratinocytes. The IC50 values for KB cells after 6 and 24 h treatment with NCTD were 209.97 and 15.06 mg/ml respectively, whereas the values for normal keratinocytes were 3865.69 and 216.29 mg/ml (Table 1). The selective indices (SI; the ratios of IC50 values of normal Table 1 IC50 valuesa (mg/ml) of NCTD on KB cells and normal keratinocytes Treatment period
KB cells
Normal keratinocytes
SIb
6h 24 h
209.97 15.06
3865.69 216.29
18.41 14.36
a b
Data represent the mean values of three separate experiments. SI, selective index, IC50 of normal keratinocytes/IC50 of KB cells.
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keratinocytes to that of KB cells) of NCTD treatment for 6 and 24 h were 18.41 and 14.36, respectively. This means KB cells were more than 14 fold more susceptible to NCTD induced cytotoxicity compared to normal keratinocytes. 3.2. NCTD induced significant anoikis and membrane blebbing in KB cells Phase-contrast microscope observations showed the morphological alterations of the cells after exposure to NCTD (Fig. 1). Untreated normal keratinocytes (Fig. 1A) and KB cells (Fig. 1D) were polygonal or
ovoid in appearances with well-defined intercellular spaces. The KB cells were more densely packed on the culture plates. Following exposure to 10 mg/ml of NCTD for 24 h, a significant number of KB cells were roundedup (Fig. 1E). In contrast, only very few keratinocytes showed round cell morphology 24 h after adding 10 mg/ ml of NCTD (Fig. 1B). Treatment of KB cells with 100 mg/ml of NCTD for 24 h resulted in about 90% of cells undergoing anoikis and membrane blebbing (Fig. 1F). In comparison, the keratinocytes showed round cell morphology in only about 30% of cells after 24 h exposure to 100 mg/ml of NCTD (Fig. 1C). Most of the keratinocytes were found attached to the culture plate.
Fig. 1. Morphological alterations in buccal keratinocytes and KB cells following exposure to NCTD for 24 h. (A) Untreated keratinocytes showing polygonal appearances, intercellular spaces are readily demonstrated (arrowheads). (B) Exposure of normal keratinocytes to 10 mg/ml of NCTD. Some cells are rounded (arrowheads) in appearance. (C) Exposure of normal keratinocytes to 100 mg/ml of NCTD. More cells are rounded-up (arrowheads) and some cells have cell membrane protrusions (arrow), however, most cells remain attached to the plate. (D) Untreated KB cells, polygonal and ovoid with well-defined intercellular spaces (arrowheads). (E) Exposure of KB cells to 10 mg/ml of NCTD. A significant number of cells are round (arrowheads). (F) Exposure of KB cells to 100 mg/ml of NCTD. Most cells are round (arrowheads) and membrane blebbing is readily noted (arrows; All200).
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3.3. NCTD reduced colony forming ability of KB cells in a dose and time-dependent manner Clonogenic survival was studied in KB cells treated with different doses of NCTD (0, 5, 10, 25 or 50 mg/ml). NCTD demonstrated a clear negative effect on colonyforming ability compared to controls (Fig. 2). NCTD treatment resulted in a dose and exposure time-dependent decline in colony counts. Significant clonogenic inhibition was noted even when the cells were exposed to a low concentration (5 mg/ml) of drug for a short period of time (6 h). 3.4. Significant growth retardation induced by NCTD in KB cells but not in keratinocytes KB cells and normal keratinocytes were treated with various concentrations of NCTD (0, 5, 10, 25 or 50 mg/ ml) for 6 and 24 h and the effect on subsequent growth of the survived cells was observed. Fig. 3 shows marked growth retardation of KB cells following exposure to NCTD. For 6 h exposure, lower concentrations (5 and 10 mg/ml) of NCTD caused significant inhibition of cell growth in the early 3 or 4 days but a delayed recovery was still noted (Fig. 3A). With higher concentrations, NCTD inhibited cell growth completely (50 mg/ml) or only a weak recovery was noted (25 mg/ml) in the later days of the experiment. When treated for a longer time (24 h), NCTD inhibited the growth of KB cells to a large extent (Fig. 3B). A delayed recovery was noted only for the lowest concentration (Fig. 3B, 5 mg/ml) and higher concentrations of NCTD (Fig. 3B, 25 mg/ml, and 50 mg/ml) resulted in almost complete inhibition of cell growth throughout the assay period (day 1–day 5). NCTD treatment for 6 and 24 h reduced the numbers of
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normal keratinocytes to a certain extent. However, the subsequent growth of the survived keratinocytes was largely not inhibited (Fig. 4). Only transient growth inhibition was noted in the buccal keratinocytes treated with up to 50 mg/ml of NCTD for 6 (Fig. 4A) or 24 h (Fig. 4B). Cell growth of the NCTD-treated keratinocytes caught up with that of the untreated controls on the third or fourth day after drug exposure. 3.5. Buccal keratinocytes were more susceptible to NCTD induced DNA synthesis inhibition The effect of NCTD on [methyl-3H]-thymidine incorporation in KB cells and normal keratinocytes were studied. NCTD inhibited de novo DNA synthesis in a dose and exposure time-dependent manner (Fig. 5). At lower concentrations (5 and 10 mg/ml), 6 h NCTD treatment inhibited DNA synthesis to a similar extent in KB cells and normal keratinocytes but the inhibition was significantly more marked in normal keratinocytes after a 24 h-exposure (Fig. 5). At higher concentrations NCTD further reduced the incorporation of 3H-thymidine and DNA synthesis was almost completely abolished in both normal keratinocytes and KB cells by 6 and 24 h exposure to 50 mg/ml of NCTD (Fig. 5). 3.6. Higher concentrations of NCTD induced significant DNA strand break in KB cells but not in keratinocytes DNA strand breaks were determined in KB cells and normal keratinocytes by DNA precipitation assay after treatment with various concentrations of NCTD. The amount of DNA released from the nucleoprotein (supernatant) was compared to that still attached to the protein (precipitate). The proportion of DNA remaining in the supernatant was a measure of DNA damage in the cells. As shown in Fig. 6, significant DNA damage was noted only in KB cells after exposing the cells to higher concentrations (510 mg/ml) of NCTD for a long period (24 h). A 6 h-exposure to NCTD resulted in no significant DNA damage in KB cells regardless of the concentrations used. NCTD caused no significant DNA damage in normal keratinocytes with the doses and exposure time used in the experiment.
4. Discussion
Fig. 2. Clonogenic assay of KB cells after NCTD treatment. The cells were treated with various concentrations of NCTD for 6 and 24 hr. The numbers of colonies (at least 50 cells) were counted after 10 days of growth. Results are expressed as percentages of colony number relative to the untreated control. Each point represents the mean of triplicate experiments. Bars, S.D. *P <0.05 vs. control (0 mg/ml).
The role of chemotherapy in treating head and neck cancer has been expanding. In recent years, concurrent chemoradiotherapy programs have resulted in clinically and statistically significant gains in the management of squamous cell carcinoma of the head and neck [4,5]. However, each program has its risks and benefits and myelosuppression remains the major limiting toxicity for most of the regimens in use today [21]. Conse-
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Fig. 3. Growth curves of KB cells after NCTD treatment. The cells were treated with various concentrations of NCTD for 6 (A) and 24 hr (B). The cells were washed and fresh medium was added. The numbers of cells were counted in a hemocytometer 0, 1, 2, 3, 4 and 5 days after drug removal. Each point represents the mean of triplicate experiments. Bars, S.D. *P<0.05 vs. control (0 mg/ml).
Fig. 4. Growth curves of normal buccal keratinocytes after NCTD treatment. The cells were treated with various concentrations of NCTD for 6 (A) and 24 hr (B). The cells were washed and fresh medium was added. The numbers of cells were counted in a hemocytometer 0, 1, 2, 3, 4 and 5 days after drug removal. Each point represents the mean of triplicate experiments. Bars, S.D. *P <0.05 vs. control (0 mg/ml).
quently, it is necessary to continue searching for cytotoxic agents to tumor cells with improved or altered efficacy profile and minimum toxicity to hematopoietic system and normal tissues. NCTD has been shown to be effective in inhibiting tumor cell proliferation in addition to its ability to stimulate the bone marrow activity [10,14]. Although it was used to treat cancers in China since 1984 [10], its cytotoxic effects on oral cancer cells has not been reported. In the present study, we showed that NCTD can cause acute toxicity to oral cancer cells. Normal oral keratinocytes are relatively resistant to NCTD treatment (SI for 6 and 24 h treatment were 18.41 and 14.36, respectively). The IC50 values of NCTD on KB cells were comparable to those determined for other tumor cell lines. McCluskey
et al. [12] recently reported that the IC50 values of various tumor cell lines derived from ovary, colon and osteosarcoma after 72 h exposure to NCTD were in the range of 20 to 50 mM. Our results showed the IC50 of KB cells after NCTD treatment for 24 h was 15.06 mg/ ml (89.6 mM). The cytotoxicity of NCTD was further demonstrated by its negative effect on the colony-forming ability of KB cells. Clonogenic inhibition was obvious even after a short exposure (6 h) to 5 mg/ml of NCTD. Cell shrinkage and membrane blebbing were observed after NCTD treatment and such alterations are compatible with the apoptotic morphology [22]. These results are comparable to those in our previous report showing NCTD induced similar morphologic changes and apoptosis in glioblastoma cells [11].
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Fig. 5. Inhibition of DNA synthesis in normal keratinocytes and KB cells by NCTD. The cells were treated with various concentrations of NCTD for 6 and 24 hr and the incorporation of 3H-thymidine was assayed. Results are expressed as percentages of 3H-thymidine incorporation relative to the untreated control. Each point represents the mean of triplicate experiments. Bars, S.D. *P<0.05 vs. control (0 mg/ ml).
The kinetics of cell growth after NCTD exposure revealed interesting differences between KB and normal keratinocytes. Normal keratinocytes could recover from the drug treatment and their growth caught up with that of the untreated cells even after exposure to 50 mg/ml of NCTD for 24 h. In contrast, significant growth retardation was noted after KB oral cancer cells treated with as low as 10 mg/ml of NCTD for 24 h. The exposure of KB cells to 50 mg/ml of NCTD for 6 or 24 h resulted in complete inhibition of subsequent cell growth (Fig. 2). These after-effects may be attributed to irreversible changes in cellular functions induced by NCTD that lead to delayed cell death. The exact mechanism of the selective cytotoxicity of NCTD on normal buccal keratinocytes and oral cancer cells needs further investigation. NCTD inhibited DNA synthesis in a dose and exposure time-dependent manner. It is interesting to find out that NCTD blocked DNA synthesis to a larger extent in normal keratinocytes than that in KB cells. The concentration resulted in 50% inhibition of 3H-thymidine incorporation in normal keratinocytes (5.70 mg/ml) was lower than the IC50 for acute cell death (216.29 mg/ml) after 24 h NCTD exposure. In KB cells, the 50% inhibitory concentration of 3H-thymidine incorporation (19.73 mg/ml) was similar to the IC50 for acute cell death (15.06 mg/ml) when treated with NCTD for 24 h. The results indicated that NCTD caused transient growth arrest in normal keratinocytes since cell growth could catch up with the untreated control several days after drug removal. The signaling pathways underlying the selection between cell death and growth arrest should be addressed by future investigations on NCTD cytotoxicity. Many of the chemotherapeutic agents in use today are genotoxic. In our study, NCTD induced DNA damage only in KB cells at higher concentrations (510 mg/ml)
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Fig. 6. DNA strand breaks in normal keratinocytes and KB cells induced by NCTD. The cells were treated with various concentrations of NCTD for 6 and 24 hr and DNA strand breaks were measured by DNA precipitation assay. Radioactivity of the supernatants (which contained DNA strand breaks) and the precipitates (which contained bulk DNA) was measured separately. The radioactivity of the precipitates was expressed as percentages of total radioactivity. Each point represents the mean of triplicate experiments. Bars, S.D. *P <0.05 vs. control (0 mg/ml) 2–4.
and longer exposure (24 h). DNA strand break was seen only after cell viability was reduced to less than 60% of the control, indicating that DNA strand break was the consequence of extragenomic damage and viability loss [23]. In conclusion, our results showed that NCTD is more effective in inducing cytotoxicity in oral cancer cells compared to normal buccal keratinocytes as demonstrated by acute cell death and cell growth inhibition assays after drug treatment. Oral cancer cells are more readily killed by NCTD and their subsequent growth is inhibited even after drug removal. On the other hand, NCTD can cause temporary growth arrest in normal keratinocytes. The differential toxicity of NCTD on oral cancer cells and buccal keratinocytes may be of value to its clinical application.
Acknowledgements This work was supported in part by grants 89-2314-B002-366 (Lin) from National Science Council and DOH89-TD-1134, DOH90-TD-1041 (Hong) from National Health Research Institute, Taiwan, ROC. References [1] Notani PN. Epidemiology and prevention of head and neck cancer: a global view. In: Saranath D, editor. Contemporary Issues in Oral Cancer. New Delhi: Oxford University Press, 2000. p. 1–29.
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