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Induction of apoptosis in KB cells by pingyangmycin
Oral Oncology 36 (2000) 242±247
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Induction of apoptosis in KB cells by pingyangmycin Kuo-Wei Taia, Ming-Yung Choua, Chao-Chin Hub, Jaw-Ji Yangc, Yu-Chao Changc,* a
Department of Oral and Maxillofacial Surgery, Chung Shan Medical and Dental College Hospital, Taichung, Taiwan, ROC b Institute of Biochemistry, Chung Shan Medical and Dental College, Taichung, Taiwan, ROC c School of Dentistry, Chung Shan Medical and Dental College, 23 Section 1, Taichung-Kang Road, Taichung, Taiwan, ROC Received 23 July 1999; accepted 1 October 1999
Abstract Pingyangmycin (PYM; Bleomycin A5), an antitumour antibiotic is currently used during anticancer therapy. Previous experiments demonstrated that the therapeutic eciency of PYM for treatment of malignant tumours is considered to be related to its ability to cause DNA strand breaks in vitro. However, very little is known about the interaction of PYM with the target cells, and it is still unclear how PYM enters the cells. In this study, cell death induced by PYM was studied in a human squamous cell carcinoma cell line (KB cells). In order to determine if cell death occurred by necrosis (reproductive cell death) or apoptosis (programmed cell death), KB cells were exposed to dierent concentrations of PYM and evaluated by biochemical and morphological criteria. Our results indicate that KB cells displayed an arrest in the G2-M phase of the cell cycle and became enlarged and polynucleated before dying at the low concentrations of PYM. In contrast, when cells were exposed to high concentrations of PYM, morphological changes identical to those usually associated with apoptosis were observed as well as internucleosomal digestion of genomic DNA. In conclusion, we demonstrate that PYM is able to induce two distinct modes of cell death depending on the doses of PYM. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Apoptosis; Pingyangmycin; Bleomycin A5; KB cell
1. Introduction Pingyangmycin (PYM; bleomycin A5), an antitumour antibiotic isolated from Streptomyces pingyangensisn cultures, has been shown to be a glycopeptide complex containing the same disaccharide moiety (l-glucose and 3-O-carbamoyl-d-mannose) and a peptide part (®ve amino acids, one amine) as bleomycin (BLM). But in its terminal amine moiety, the spermidine derivative (A5) is the major component [1±3]. Although PYM is used clinically to treat a variety of tumors [4,5], the action mechanism of PYM has been poorly studied. Recently, we found that PYM has a cytocidal activity against cultured tumour cells, the mechanism of PYM-induced cytotoxicity is considered related to its ability to inhibit DNA systhesis and cause DNA strand breaks in vitro [6]. However, the interaction of PYM with whole cells and how PYM enters the cytoplasm is still not fully understood.
* Corresponding author. Tel.: +886-4-2015111, ext. 6268; fax: +886-4-2024349.
There are two major morphologically and biochemically distinct processes whereby death occurs in eukaryotic cells: necrosis (reproductive cell death) and apoptosis (programmed cell death) [7]. The morphological changes associated with necrosis are cell swelling, rupture of membranes, and lysis of organized structure. In contrast, during the process of apoptosis, condensation and fragmentation of the cytoplasm and nucleus occur in the target cells while normal organelle structure is maintained. Biochemically, necrosis results from loss of osmoregulation, with random DNA digestion by lysosomal enzymes at a late stage. During apoptosis, internucleosomal DNA digestion is caused by the activation of an endogenous endonuclease, which is proposed to play a key role in apoptosis [8]. Previous study has indicated that many chemotherapeutic drugs could induce apoptosis in a variety of tumour cells [9,10]. According to Tounekti et al. [11], BLM can induce two distinct modes of cell death. The mechanism of the cell death is closely related to the number of BLM molecules introduced into the cell [12]. At high concentrations, BLM can be considered as an apoptosis mimetic. To date there has been very little research concerned about
1368-8375/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S1368-8375(99)00086-X
Kuo-Wei Tai et al. / Oral Oncology 36 (2000) 242±247
243
PYM-induced cell death, we tried to analyze whether the mode of cell death can be aected by simply increasing PYM concentrations or not. In this study, we demonstrate that PYM can induce two types of cell death of KB cells depending on the concentrations of PYM. We employed a modi®ed gel electrophoresis procedure which works without timeconsuming organic extractions and provides rapid visualization of the characteristic nucleosome ladder of apoptosis [13]. We also investigated the other elements of apoptosis by ¯ow cytometry and morphologic observation. Once apoptosis was established as a mechanism for cell death, cell cycle analysis was performed to determine if cells arrested in a particular stage of the cell cycle.
were plated 2105 cells per well into six-well plates (Falcon). After overnight attachment, cells were divided two groups. The cells of group one were exposed to 5 mg/ml PYM for various incubation times (4, 12, 24 h) and the other was treated with dierent doses of PYM (5, 50, 500 mg/ml, and 5 mg/ml) for 24 h. Then the ¯oating cells in the medium were combined with attached cells harvested by trypsinization. Cells were washed with cold PBS and ®xed in 80% ethanol in PBS at ÿ20 C. After 12 h, ®xed cells were pelleted, gently resuspended in cold PBS and supplemented with 0.5 mg/ml propidium iodide plus 50 mg/ml RNase A (Sigma). Samples were then incubated at 37 C for 30 min, stored in the dark at 4 C, and analyzed on a Becton Dickinson FACScan ¯ow cytometer.
2. Materials and methods
2.4. Morphology analysis
2.1. Cells and chemicals
The morphology of KB cells assessed as described by Sugimoto et al. [17] was observed by Giemsa staining to dierentiate necrosis from apoptosis. After treatment with dierent concentrations of PYM for 24 h, cells were ®xed with methanol (Merck, Germany) for 10 min and stained in Giemsa solution (Merck) for 20 min. The morphology of KB cells were observed under a Nikon phase-contrast microscope and documented by photography. When the cells showed the feature of necrosis, cell swelling and rupture of membrane might be seen. When that of apoptosis, cell shrinkage and condensation of the cytoplasm should be observed.
KB cells, human squamous cell carcinoma cell line (ATCC CCL-17; American Type Culture Collection, Rockville, MD, USA) were grown in Dulbecco's Modi®ed Eagle medium (DMEM; Gibco, Grand Island, NY, USA), supplemented with 10 fetal bovine serum (Gibco), penicillin, and streptomycin. Cells were cultivated as monolayers in plastic culture ¯asks (Nunc, Denmark) at 37 C under a humidi®ed atmosphere of 5% CO2 in air. PYM hydrochloride was purchased from Hebei pharmaceutical factory (Tianjin, People's Republic of China); appropriate concentrations range of 0.5 mg/ml± 5 mg/ml were set up in DMEM. 2.2. Cytotoxicity assay KB cells survival was determined by MTT assays, as described [14]. Brie¯y, the tetrazolium salt MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma, St. Louis, MO, USA) solution was prepared as 5 mg/ml in phosphate-buered saline (PBS; Gibco) just before use. KB cells were diluted in fresh complete medium and seeded in 24-well plates (Falcon, Franklin Lakes, NJ, USA) (5104 cells/well). After 24 h incubation time, cells were treated with various concentrations of PYM (0.5, 5, 50, 500 mg/ml, and 5 mg/ml) for 24 h, then 200 ml MTT dye was added to each well. The plates were incubated in a CO2 incubator for 4 h. Optical density was determined by eluting the dye with isopropanol/formic acid, and the spectrophotometric absorbance was measured at 563 nm using a spectrophotometer (Hitachi, Japan). 2.3. Cell cycle analysis Cell cycle analysis was performed on cells stained with propidium iodine as previous detailed [15,16]. KB cells
2.5. DNA fragmentation analysis by gel electrophoresis In order to isolate apoptotis DNA fragments from treated cells, the method was according to Herrmann et al. [13]. Exponentially growing cells were seeded in sixwell plates (2105 cells/well). After 24 h, cells were treated with dierent concentrations of PYM (5, 50, 500 mg/ml, and 5 mg/ml) for 24, 48, and 72h. The cells were harvested and washed in cold PBS and pelleted by centrifugation. Cell pellets were then lysed for 10 s with 50 ml lysis buer (1% NP 40 in 20 mM EDTA, 50 mM Tris±HCl, pH 7.5). After centrifugation at 1600 g for 5 min, the supernatant was collected and extraction was repeated with the same amount of lysis buer. Apoptotic DNA fragments were recovered from the supernanants by adding sodium dodecyl sulphate (BioRad, Richmond, CA, USA) to 1% and treating for 2 h with RNase A (®nal concentration 5 mg/ml) at 56 C followed by digestion with proteinase K (®nal concentration 2.5 mg/ml) for 2 h at 37 C. DNA was precipitated with 0.5 vol. 10 M ammonium acetate and 2.5 vol. ethanol, dissoved in gel loading buer and separated by electrophoresis in 1% agarose gels. Molecular size markers, both a 200-base pair (Gibco) and a l/HindIII ladder (Gibco), were included in the gel for comparison. After
244
Kuo-Wei Tai et al. / Oral Oncology 36 (2000) 242±247
the gel electrophoresis, the DNA framents in the gel were stained with ethidium bromide and visualized by UV illumination for ladder formation. 2.6. Statistical analysis Three replicates of each concentration were performed in each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was performed by one-way analysis of variance. Means and standard deviations were calculated. Probability values of p<0.05 were regarded as statistically signi®cant.
KB cells in the absence of PYM display normal pro®le with a peak at 84.3 units of ¯uorescence representing cells in G0-G1 and a peak at 146.7 units of ¯uorescence representing G2-M (Fig. 3b). Flow cytometric analysis of KB cells treated with 5 mg/ml PYM revealed a subpopulation displaying propidium iodine ¯uorescence reduced compared to that of the G0-G1 cell cycle region which can be considered as the A0 region described by
3. Result The MTT assay is a colorimetric assay based on the ability of the viable cells to reduce a soluble yellow tetrazolium salt (MTT) to blue formazan crystals. As can be seen in Fig. 1, PYM had a cytotoxic eect on KB cells in a concentration-dependent manner. The viability of KB cells was decreased to 98.95, 84.45, 74.95, 47.58, and 30.83% at the concentrations of 0.5, 5, 50, 500 mg/ ml, and 5 mg/ml. Cell viability was obviously inhibited at the concentration of 5 mg/ml PYM for 24 h ( p<0.05). Fig. 2 presents the eect of 5 mg/ml PYM on the cell cycle of KB cells. As shown by ¯ow cytometric analysis, cells displayed a marked arrest in the G2-M phase of the cell cycle. In addition, KB cells exposed to PYM at a dose range of 5 mg/ml±5 mg/ml during longer incubation time (24 h) were also investigated by ¯ow cytometry.
Fig. 1. Cytotoxicity assay with MTT staining of viable cells. KB cells were treated with dierent concentrations of pingyangmycin (PYM). When treated with 5 mg/ml PYM, the cell viability is obviously inhibited ( p<0.05).
Fig. 2. Flow cytometric DNA analysis of KB cells at various times in the presence of 5 mg/ml pingyangmycin (PYM). The number of cells is represented as a function of ¯uorescence.
Kuo-Wei Tai et al. / Oral Oncology 36 (2000) 242±247
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Fig. 3. Flow cytometric DNA analysis of KB cells treated with dierent concentrations of pingyangmycin (PYM). The number of cells is represented as a function of ¯uorescence. (a) Cells treated with 5 mg/ml for 24 h. Notice the A0 peak (¯uorescence 48.5) characteristic of apoptotic cells. (b) Cells in the absence of PYM display normal pro®le with a peak at 84.3 units of ¯uorescence representing cells in G0-G1 and a peak at 146.7 units of ¯uorescence representing in G2-M.
Telford et al. [18] in cell populations undergoing apoptosis (Fig. 3a). Morphological changes in KB cells treated with various doses of PYM were observed by Giemsa staining to dierentiate necrosis from apoptosis (Fig. 4). Within 24 h of exposure to 50 mg/ml PYM, most cells showed features of necrosis. Cells were enlarged compared to the untreated cells, some were multinucleated, while others displayed an arrest in mitosis. These results show signs of a highly disturbed cell cycle (Fig. 4b). However, when the cells were treated with 5 mg/ml PYM, the number of apoptotic cells was obviously more than necrotic cells. These cells exhibited no increase in size but on the contrary marked shrinkage, membrane blebbing and condensation of the cytoplasm (Fig. 4c). Fig. 5 shows the time course of DNA fragmentation in KB cells with the treatment of various concentrations of PYM. We detected the oligonucleosomal ladder characteristic of apoptosis by modi®ed agarose gel electrophoresis. No ladder bands were detected when PYM concentrations were below 50 mg/ml whatever the time (24, 48, 72 h) and in control cells. After incubation for 24 h, DNA ladder bands were weakly detectable by the treatment of 500 mg/ml and were clearly observed by the treatment of 5 mg/ml. The characteristic pattern of DNA fragments consisted of multimers of approximately 180 base pairs. These digestion products correspond to the nuleosome ladders characteristic of apoptosis 4. Discussion Chemotherapy for head and neck cancer has been advanced by the development of a variety of eective
anticancer agents. Understanding of the cellular processes involved and the molecular mechanisms responsible for the cell death induced by anticancer drugs may aid in the development of more eective and tumourselective chemotherapy [9,10,17,19]. In China, the antitumour antibiotic PYM is fairly extensively used in chemotherapy for treatment of squamous cell carcinoma, malignant lymphoma, Hodgkin's disease, and lymphangioma, the common benign tumour found most frequently in the head and neck [4,5,20,21]. Our previous study has shown that 5 mg/ml PYM could inhibit cell growth and induce DNA strand breaks [6]. In the present study, PYM inhibited cell viability in a dosedependent manner. These results have clearly shown the cytotoxicity of PYM. Our study shows that PYM induced two distinct modes of cell death. We used a single cell line in this study since it was likely that dierent cell lines may have inherently dierent mechanisms for the induction of apoptosis and for the intracellular transduction of apoptotic signals [22]. KB cells became enlarged, multinucleated, and displayed an arrest in G2-M phase of the cell cycle in the presence of low concentrations of PYM (5, 50 mg/ml). Even after very long incubation time (72 h), only a smear, resulting from random DNA degradation can be detected. In contrast, KB cells exhibited the morphological and biochemical changes associated with apoptosis when in the presence of high concentrations of PYM (500 mg/ml, 5 mg/ml). The morphologic changes such as cell shrinkage and chromatin condensation are observed as well as DNA fragmentation into oligonucleosomal-sized fragments. Simply increasing PYM concentrations can aect the mode of cell death. The mode of cell death changes from necrosis to
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Kuo-Wei Tai et al. / Oral Oncology 36 (2000) 242±247
Fig. 5. Time course of DNA fragmentation in KB cells. Lanes A±E, cells treated with dierent concentrations of PYM (0, 5, 50, 500 mg/ml, and 5 mg/ml) for 24 h; lanes F±J, cells treated with dierent concentrations of PYM (0, 5, 50, 500 mg/ml, and 5 mg/ml) for 48 h; lanes K±O, cells treated with dierent concentrations of PYM (0, 5, 50, 500 mg/ml, and 5 mg/m1) for 72 h; l, l/Hind III; M, 200 bp ladder.
Fig. 4. The morphological variations of KB cells treated with various concentrations of pingyangmycin (PYM) for 24 h were observed (400). (a) Untreated cells; (b) cells treated with 50 mg/m1 PYM; (c) cells treated with 5 mg/m1 PYM. Small arrows indicate apoptotic cells, and large arrows, necrotic cells.
apoptosis can be considered [23]. Although the mechanism of PYM uptake is not clear, it is implied that PYM might cross the cell membrane by slow passive diusion. According to our results, some characteristics of PYM can be described: (1) only a very little amount of the external PYM enters the intact cells; the plasma membrane is a strongly limiting step for the access to
their intracellular target; (2) PYM is already ecient on intact cells at low concentrations because it has a very high intrinsic toxicity; and (3) PYM acts directly as an endonuclease and can be considered as an apoptosismimetic drug at high concentrations. Until our present study, the relationship between the number of PYM molecules and their biological eects had never been studied. Our results support the hypothesis that the PYM-generated DNA strand breaks are responsible for PYM cytotoxicity. Moreover, the endonuclease ability of PYM results in two mechanisms of cell death which are closely related to the number of PYM molecules present inside the cell, and to the number of DNA strand breaks generated as described by our earlier report [6]. From a clinical viewpoint, it might be very signi®cant for cancer chemotherapy if we were able to speci®cally induce tumour cell apoptosis. Apoptotic cell death is not associated with the release of intracellular constituents into body ¯uids upon cell destruction, as occurs with necrotic cell death, and so it could prevent secondary tissue damage due to in¯ammatory reactions [23]. However, our results indicate that in order to induce apoptosis, the concentration of PYM present in the culture medium is too high for clinical use. Therefore, how to increase cell membrane permeability to allow more PYM molecules into the target cell easily will be the next subject for us to study. Acknowledgement This study was supported by Chung Shan Medical and Dental College grants CSMC 88-OM-B-010.
Kuo-Wei Tai et al. / Oral Oncology 36 (2000) 242±247
References [1] Xue SB, Cheng ZH, Liu HT, Li SW, Zhang HQ, Cheng RX, Hu YY, Xu P. Eects of bleomycin A5 on cell cycle of Chinese hamster ovary cells. Chung Kuo Yao Li Hsueh Pao 1984;5:112± 4(in Chinese). [2] Umezawa H, Maeda K, Takeuchi T, Okami Y. New antibiotics, bleomycin A and B. Journal of Antibiotic 1966;19:200±9. [3] Umezawa H, Takeuchi T, Hori S, Sawa T, Ishizuka M. Studies on the mechanism of antitumor eect of bleomycin on squamous cell carcinoma. Journal of Antibiotic 1972;25:409±20. [4] He NG, Zhang HQ, Song PG, Liu ZM, Xue SB. Mechanism of enhancement of bleomycin A5 antitumor activity by verapamil. Yao Hsueh Hsueh Pao 1991;26:15±19(in Chinese). [5] Li A, Wang T, Yang R. Pingyangmycin-induced chromosome damage in lymphocytes of laryngeal cancer patients. Chung Hua Erh Pi Yen Hou Ko Tsa Chih 1994;29:333±5(in Chinese). [6] Tai KW, Chang YC, Chou LSS, Chou MY. Cytotoxicity eect of pingyangmycin on cultures KB cells. Oral Oncology 1998;34: 219±23. [7] Wyllie AH, Kerr JFR, Currie AR. Cell death: the signi®cance of apoptosis. International Review of Cytology 1980;68:251±356. [8] Arends MJ, Morris RG, Wyllie AH. Apoptosis: the role of the endonuclease. American Journal of Pathology 1990;136:593±608. [9] Barry MA, Belinke CA, Eastman A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthemia. Biochemical Pharmacology 1990;40:2353±62. [10] Barry MA, Reynolds JE, Eastman A. Etoposide-induced apoptosis in human HL-1360 cells is associated with intracellular acidi®cation. Cancer Research 1993;53:2349±57. [11] Tounekti O, Pron G, Belehradek J, Mir LM. Bleomycin, an apoptosis-mimetic drug that induces two types of cell death depending on the number of molecules internalized. Cancer Research 1993;53:5462±9. [12] Orlowski S, Belehradek Jr. J, Paoletti C, Mir LM. Transient electropermeabilization of cells in culture. Biochemical Pharmacology 1988;37:4727±33.
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[13] Herrmann M, Lorenz HM, Voll R, GruÈnke K, Woith W, Kalden JR. A rapid and simple method for the isolation of apoptotic DNA fragments. Nucleic Acids Research 1994;22:5506±7. [14] Araki T, Enokido Y, Inamura N, Aizawa S, Reed JC, Hatanaka H. Changes in c-Jun but not Bcl-2 family proteins in p53-dependent apoptosis of mouse cerebellar granule neurons induced by DNA damaging agent bleomycin. Brain Research 1998;794:239±47. [15] Sorenson CM, Eastman A. Mechanism of cis-diamminedichloroplatinum (II)-induced cytotoxicity: role of G2 arrest and DNA double-strand breaks. Cancer Research 1988;48:4484±8. [16] Morris SM, Domon OE, McGarrity LJ, Chen JJ, Manjanatha MG, Andrews AM, Aidoo A, Casciano DA. A role for apoptosis in the toxicity and mutagenicity of bleomycin in AHH-1 tk+/ÿ human lymphoblastoid cells. Mutation Research 1996;357:143±65. [17] Sugimoto C, Matsukawa S, Fujieda S, Noda I, Tanaka N, Tsuzuki H, Saito H. Involvement of intracellular glutathione in induction of apoptosis by cisplatin in a human pharyngeal carcinoma cell line. Anticancer Research 1996;16:675±80. [18] Telford WG, King LE, Fraker PJ. Evaluation of glucocorticoidinduced DNA fragmentation in mouse thymocytes by ¯ow cytornetry. Cell Proliferation 1991;24:447±59. [19] MuÈller M, Strand S, Hug H, Heinemann EM, Walczak H, Homan WJ, Stremmel W, Krammer PH, Galle PR. Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/ Fas) receptor/ligand system and involves activation of wild-type p53. Journal of Clinical Investigation 1997;99:403±13. [20] Zhong PQ, Zhi FX, Li R, Xue JL, Shu GY. Long-term results of intratumorous bleomycin-A5 injection for head and neck lymphangioma. Oral Surgery, Oral Medicine, Oral Pathology 1998;86:139±44. [21] Wang SM. Prevention and treatment of pulmonary toxicity reactions of PYM (8-case report). Chinese Cancer 1989;8:36±8. [22] McConkey DJ, Orrenius S. Signal transduction pathways to apoptosis. Trends Cell Biology 1994;4:370±5. [23] Fernandes RS, Cotter TG. Apoptosis or necrosis: intracellular levels of glutathione in¯uence mode of cell death. Biochemical Pharmacology 1994;48:675±81.
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Induction of apoptosis in KB cells by pingyangmycin Kuo-Wei Taia, Ming-Yung Choua, Chao-Chin Hub, Jaw-Ji Yangc, Yu-Chao Changc,* a
Department of Oral and Maxillofacial Surgery, Chung Shan Medical and Dental College Hospital, Taichung, Taiwan, ROC b Institute of Biochemistry, Chung Shan Medical and Dental College, Taichung, Taiwan, ROC c School of Dentistry, Chung Shan Medical and Dental College, 23 Section 1, Taichung-Kang Road, Taichung, Taiwan, ROC Received 23 July 1999; accepted 1 October 1999
Abstract Pingyangmycin (PYM; Bleomycin A5), an antitumour antibiotic is currently used during anticancer therapy. Previous experiments demonstrated that the therapeutic eciency of PYM for treatment of malignant tumours is considered to be related to its ability to cause DNA strand breaks in vitro. However, very little is known about the interaction of PYM with the target cells, and it is still unclear how PYM enters the cells. In this study, cell death induced by PYM was studied in a human squamous cell carcinoma cell line (KB cells). In order to determine if cell death occurred by necrosis (reproductive cell death) or apoptosis (programmed cell death), KB cells were exposed to dierent concentrations of PYM and evaluated by biochemical and morphological criteria. Our results indicate that KB cells displayed an arrest in the G2-M phase of the cell cycle and became enlarged and polynucleated before dying at the low concentrations of PYM. In contrast, when cells were exposed to high concentrations of PYM, morphological changes identical to those usually associated with apoptosis were observed as well as internucleosomal digestion of genomic DNA. In conclusion, we demonstrate that PYM is able to induce two distinct modes of cell death depending on the doses of PYM. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Apoptosis; Pingyangmycin; Bleomycin A5; KB cell
1. Introduction Pingyangmycin (PYM; bleomycin A5), an antitumour antibiotic isolated from Streptomyces pingyangensisn cultures, has been shown to be a glycopeptide complex containing the same disaccharide moiety (l-glucose and 3-O-carbamoyl-d-mannose) and a peptide part (®ve amino acids, one amine) as bleomycin (BLM). But in its terminal amine moiety, the spermidine derivative (A5) is the major component [1±3]. Although PYM is used clinically to treat a variety of tumors [4,5], the action mechanism of PYM has been poorly studied. Recently, we found that PYM has a cytocidal activity against cultured tumour cells, the mechanism of PYM-induced cytotoxicity is considered related to its ability to inhibit DNA systhesis and cause DNA strand breaks in vitro [6]. However, the interaction of PYM with whole cells and how PYM enters the cytoplasm is still not fully understood.
* Corresponding author. Tel.: +886-4-2015111, ext. 6268; fax: +886-4-2024349.
There are two major morphologically and biochemically distinct processes whereby death occurs in eukaryotic cells: necrosis (reproductive cell death) and apoptosis (programmed cell death) [7]. The morphological changes associated with necrosis are cell swelling, rupture of membranes, and lysis of organized structure. In contrast, during the process of apoptosis, condensation and fragmentation of the cytoplasm and nucleus occur in the target cells while normal organelle structure is maintained. Biochemically, necrosis results from loss of osmoregulation, with random DNA digestion by lysosomal enzymes at a late stage. During apoptosis, internucleosomal DNA digestion is caused by the activation of an endogenous endonuclease, which is proposed to play a key role in apoptosis [8]. Previous study has indicated that many chemotherapeutic drugs could induce apoptosis in a variety of tumour cells [9,10]. According to Tounekti et al. [11], BLM can induce two distinct modes of cell death. The mechanism of the cell death is closely related to the number of BLM molecules introduced into the cell [12]. At high concentrations, BLM can be considered as an apoptosis mimetic. To date there has been very little research concerned about
1368-8375/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S1368-8375(99)00086-X
Kuo-Wei Tai et al. / Oral Oncology 36 (2000) 242±247
243
PYM-induced cell death, we tried to analyze whether the mode of cell death can be aected by simply increasing PYM concentrations or not. In this study, we demonstrate that PYM can induce two types of cell death of KB cells depending on the concentrations of PYM. We employed a modi®ed gel electrophoresis procedure which works without timeconsuming organic extractions and provides rapid visualization of the characteristic nucleosome ladder of apoptosis [13]. We also investigated the other elements of apoptosis by ¯ow cytometry and morphologic observation. Once apoptosis was established as a mechanism for cell death, cell cycle analysis was performed to determine if cells arrested in a particular stage of the cell cycle.
were plated 2105 cells per well into six-well plates (Falcon). After overnight attachment, cells were divided two groups. The cells of group one were exposed to 5 mg/ml PYM for various incubation times (4, 12, 24 h) and the other was treated with dierent doses of PYM (5, 50, 500 mg/ml, and 5 mg/ml) for 24 h. Then the ¯oating cells in the medium were combined with attached cells harvested by trypsinization. Cells were washed with cold PBS and ®xed in 80% ethanol in PBS at ÿ20 C. After 12 h, ®xed cells were pelleted, gently resuspended in cold PBS and supplemented with 0.5 mg/ml propidium iodide plus 50 mg/ml RNase A (Sigma). Samples were then incubated at 37 C for 30 min, stored in the dark at 4 C, and analyzed on a Becton Dickinson FACScan ¯ow cytometer.
2. Materials and methods
2.4. Morphology analysis
2.1. Cells and chemicals
The morphology of KB cells assessed as described by Sugimoto et al. [17] was observed by Giemsa staining to dierentiate necrosis from apoptosis. After treatment with dierent concentrations of PYM for 24 h, cells were ®xed with methanol (Merck, Germany) for 10 min and stained in Giemsa solution (Merck) for 20 min. The morphology of KB cells were observed under a Nikon phase-contrast microscope and documented by photography. When the cells showed the feature of necrosis, cell swelling and rupture of membrane might be seen. When that of apoptosis, cell shrinkage and condensation of the cytoplasm should be observed.
KB cells, human squamous cell carcinoma cell line (ATCC CCL-17; American Type Culture Collection, Rockville, MD, USA) were grown in Dulbecco's Modi®ed Eagle medium (DMEM; Gibco, Grand Island, NY, USA), supplemented with 10 fetal bovine serum (Gibco), penicillin, and streptomycin. Cells were cultivated as monolayers in plastic culture ¯asks (Nunc, Denmark) at 37 C under a humidi®ed atmosphere of 5% CO2 in air. PYM hydrochloride was purchased from Hebei pharmaceutical factory (Tianjin, People's Republic of China); appropriate concentrations range of 0.5 mg/ml± 5 mg/ml were set up in DMEM. 2.2. Cytotoxicity assay KB cells survival was determined by MTT assays, as described [14]. Brie¯y, the tetrazolium salt MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma, St. Louis, MO, USA) solution was prepared as 5 mg/ml in phosphate-buered saline (PBS; Gibco) just before use. KB cells were diluted in fresh complete medium and seeded in 24-well plates (Falcon, Franklin Lakes, NJ, USA) (5104 cells/well). After 24 h incubation time, cells were treated with various concentrations of PYM (0.5, 5, 50, 500 mg/ml, and 5 mg/ml) for 24 h, then 200 ml MTT dye was added to each well. The plates were incubated in a CO2 incubator for 4 h. Optical density was determined by eluting the dye with isopropanol/formic acid, and the spectrophotometric absorbance was measured at 563 nm using a spectrophotometer (Hitachi, Japan). 2.3. Cell cycle analysis Cell cycle analysis was performed on cells stained with propidium iodine as previous detailed [15,16]. KB cells
2.5. DNA fragmentation analysis by gel electrophoresis In order to isolate apoptotis DNA fragments from treated cells, the method was according to Herrmann et al. [13]. Exponentially growing cells were seeded in sixwell plates (2105 cells/well). After 24 h, cells were treated with dierent concentrations of PYM (5, 50, 500 mg/ml, and 5 mg/ml) for 24, 48, and 72h. The cells were harvested and washed in cold PBS and pelleted by centrifugation. Cell pellets were then lysed for 10 s with 50 ml lysis buer (1% NP 40 in 20 mM EDTA, 50 mM Tris±HCl, pH 7.5). After centrifugation at 1600 g for 5 min, the supernatant was collected and extraction was repeated with the same amount of lysis buer. Apoptotic DNA fragments were recovered from the supernanants by adding sodium dodecyl sulphate (BioRad, Richmond, CA, USA) to 1% and treating for 2 h with RNase A (®nal concentration 5 mg/ml) at 56 C followed by digestion with proteinase K (®nal concentration 2.5 mg/ml) for 2 h at 37 C. DNA was precipitated with 0.5 vol. 10 M ammonium acetate and 2.5 vol. ethanol, dissoved in gel loading buer and separated by electrophoresis in 1% agarose gels. Molecular size markers, both a 200-base pair (Gibco) and a l/HindIII ladder (Gibco), were included in the gel for comparison. After
244
Kuo-Wei Tai et al. / Oral Oncology 36 (2000) 242±247
the gel electrophoresis, the DNA framents in the gel were stained with ethidium bromide and visualized by UV illumination for ladder formation. 2.6. Statistical analysis Three replicates of each concentration were performed in each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was performed by one-way analysis of variance. Means and standard deviations were calculated. Probability values of p<0.05 were regarded as statistically signi®cant.
KB cells in the absence of PYM display normal pro®le with a peak at 84.3 units of ¯uorescence representing cells in G0-G1 and a peak at 146.7 units of ¯uorescence representing G2-M (Fig. 3b). Flow cytometric analysis of KB cells treated with 5 mg/ml PYM revealed a subpopulation displaying propidium iodine ¯uorescence reduced compared to that of the G0-G1 cell cycle region which can be considered as the A0 region described by
3. Result The MTT assay is a colorimetric assay based on the ability of the viable cells to reduce a soluble yellow tetrazolium salt (MTT) to blue formazan crystals. As can be seen in Fig. 1, PYM had a cytotoxic eect on KB cells in a concentration-dependent manner. The viability of KB cells was decreased to 98.95, 84.45, 74.95, 47.58, and 30.83% at the concentrations of 0.5, 5, 50, 500 mg/ ml, and 5 mg/ml. Cell viability was obviously inhibited at the concentration of 5 mg/ml PYM for 24 h ( p<0.05). Fig. 2 presents the eect of 5 mg/ml PYM on the cell cycle of KB cells. As shown by ¯ow cytometric analysis, cells displayed a marked arrest in the G2-M phase of the cell cycle. In addition, KB cells exposed to PYM at a dose range of 5 mg/ml±5 mg/ml during longer incubation time (24 h) were also investigated by ¯ow cytometry.
Fig. 1. Cytotoxicity assay with MTT staining of viable cells. KB cells were treated with dierent concentrations of pingyangmycin (PYM). When treated with 5 mg/ml PYM, the cell viability is obviously inhibited ( p<0.05).
Fig. 2. Flow cytometric DNA analysis of KB cells at various times in the presence of 5 mg/ml pingyangmycin (PYM). The number of cells is represented as a function of ¯uorescence.
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Fig. 3. Flow cytometric DNA analysis of KB cells treated with dierent concentrations of pingyangmycin (PYM). The number of cells is represented as a function of ¯uorescence. (a) Cells treated with 5 mg/ml for 24 h. Notice the A0 peak (¯uorescence 48.5) characteristic of apoptotic cells. (b) Cells in the absence of PYM display normal pro®le with a peak at 84.3 units of ¯uorescence representing cells in G0-G1 and a peak at 146.7 units of ¯uorescence representing in G2-M.
Telford et al. [18] in cell populations undergoing apoptosis (Fig. 3a). Morphological changes in KB cells treated with various doses of PYM were observed by Giemsa staining to dierentiate necrosis from apoptosis (Fig. 4). Within 24 h of exposure to 50 mg/ml PYM, most cells showed features of necrosis. Cells were enlarged compared to the untreated cells, some were multinucleated, while others displayed an arrest in mitosis. These results show signs of a highly disturbed cell cycle (Fig. 4b). However, when the cells were treated with 5 mg/ml PYM, the number of apoptotic cells was obviously more than necrotic cells. These cells exhibited no increase in size but on the contrary marked shrinkage, membrane blebbing and condensation of the cytoplasm (Fig. 4c). Fig. 5 shows the time course of DNA fragmentation in KB cells with the treatment of various concentrations of PYM. We detected the oligonucleosomal ladder characteristic of apoptosis by modi®ed agarose gel electrophoresis. No ladder bands were detected when PYM concentrations were below 50 mg/ml whatever the time (24, 48, 72 h) and in control cells. After incubation for 24 h, DNA ladder bands were weakly detectable by the treatment of 500 mg/ml and were clearly observed by the treatment of 5 mg/ml. The characteristic pattern of DNA fragments consisted of multimers of approximately 180 base pairs. These digestion products correspond to the nuleosome ladders characteristic of apoptosis 4. Discussion Chemotherapy for head and neck cancer has been advanced by the development of a variety of eective
anticancer agents. Understanding of the cellular processes involved and the molecular mechanisms responsible for the cell death induced by anticancer drugs may aid in the development of more eective and tumourselective chemotherapy [9,10,17,19]. In China, the antitumour antibiotic PYM is fairly extensively used in chemotherapy for treatment of squamous cell carcinoma, malignant lymphoma, Hodgkin's disease, and lymphangioma, the common benign tumour found most frequently in the head and neck [4,5,20,21]. Our previous study has shown that 5 mg/ml PYM could inhibit cell growth and induce DNA strand breaks [6]. In the present study, PYM inhibited cell viability in a dosedependent manner. These results have clearly shown the cytotoxicity of PYM. Our study shows that PYM induced two distinct modes of cell death. We used a single cell line in this study since it was likely that dierent cell lines may have inherently dierent mechanisms for the induction of apoptosis and for the intracellular transduction of apoptotic signals [22]. KB cells became enlarged, multinucleated, and displayed an arrest in G2-M phase of the cell cycle in the presence of low concentrations of PYM (5, 50 mg/ml). Even after very long incubation time (72 h), only a smear, resulting from random DNA degradation can be detected. In contrast, KB cells exhibited the morphological and biochemical changes associated with apoptosis when in the presence of high concentrations of PYM (500 mg/ml, 5 mg/ml). The morphologic changes such as cell shrinkage and chromatin condensation are observed as well as DNA fragmentation into oligonucleosomal-sized fragments. Simply increasing PYM concentrations can aect the mode of cell death. The mode of cell death changes from necrosis to
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Fig. 5. Time course of DNA fragmentation in KB cells. Lanes A±E, cells treated with dierent concentrations of PYM (0, 5, 50, 500 mg/ml, and 5 mg/ml) for 24 h; lanes F±J, cells treated with dierent concentrations of PYM (0, 5, 50, 500 mg/ml, and 5 mg/ml) for 48 h; lanes K±O, cells treated with dierent concentrations of PYM (0, 5, 50, 500 mg/ml, and 5 mg/m1) for 72 h; l, l/Hind III; M, 200 bp ladder.
Fig. 4. The morphological variations of KB cells treated with various concentrations of pingyangmycin (PYM) for 24 h were observed (400). (a) Untreated cells; (b) cells treated with 50 mg/m1 PYM; (c) cells treated with 5 mg/m1 PYM. Small arrows indicate apoptotic cells, and large arrows, necrotic cells.
apoptosis can be considered [23]. Although the mechanism of PYM uptake is not clear, it is implied that PYM might cross the cell membrane by slow passive diusion. According to our results, some characteristics of PYM can be described: (1) only a very little amount of the external PYM enters the intact cells; the plasma membrane is a strongly limiting step for the access to
their intracellular target; (2) PYM is already ecient on intact cells at low concentrations because it has a very high intrinsic toxicity; and (3) PYM acts directly as an endonuclease and can be considered as an apoptosismimetic drug at high concentrations. Until our present study, the relationship between the number of PYM molecules and their biological eects had never been studied. Our results support the hypothesis that the PYM-generated DNA strand breaks are responsible for PYM cytotoxicity. Moreover, the endonuclease ability of PYM results in two mechanisms of cell death which are closely related to the number of PYM molecules present inside the cell, and to the number of DNA strand breaks generated as described by our earlier report [6]. From a clinical viewpoint, it might be very signi®cant for cancer chemotherapy if we were able to speci®cally induce tumour cell apoptosis. Apoptotic cell death is not associated with the release of intracellular constituents into body ¯uids upon cell destruction, as occurs with necrotic cell death, and so it could prevent secondary tissue damage due to in¯ammatory reactions [23]. However, our results indicate that in order to induce apoptosis, the concentration of PYM present in the culture medium is too high for clinical use. Therefore, how to increase cell membrane permeability to allow more PYM molecules into the target cell easily will be the next subject for us to study. Acknowledgement This study was supported by Chung Shan Medical and Dental College grants CSMC 88-OM-B-010.
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