臺灣博碩士論文加值系統

蜂膠主成份之一的咖啡酸苯乙酯(caffeic acid phenethyl ester, 以下簡稱CAPE)，己被證實具有抗氧化及毒殺腫瘤細胞等生物活性及藥理作用。本研究之目的即在探討CAPE所導致腫瘤細胞的死亡型態及其與更動氧化還原狀態之關聯，並探討CAPE對放射致敏作用之機轉。
首先使用CAPE處理人類白血病HL-60細胞，發現其可快速進入細胞內，並能隨著濃度及時間增加而愈能抑制HL-60細胞存活與增生。CAPE (6 ug/mL)處理HL-60細胞2天後，可抑制70.3 +/- 4.0%的細胞增生。但若預先給予N-acetyl-L-cysteine (NAC)處理，則可部分阻斷CAPE對HL-60細胞的增生抑制作用。CAPE處理導致此細胞典型的凋亡變化，包括出現DNA斷裂及形態特徵。細胞凋亡率在前述劑量下可在第三天達66.7 +/- 2.0%，CAPE (6 ug/mL)處理HL-60細胞可在6小時後降低粒線體跨膜電位達一半，並使細胞內還原態glutathione (GSH)含量在1小時後即降低至未處理組的41.7 +/- 6.0%。預先給予NAC處理可完全阻斷CAPE降低GSH之作用，並可避免部分細胞之凋亡。在活性氧化物方面，本研究發現CAPE可在2小時內快速清除細胞內過氧化氫含量達處理前的19%，但是對超氧化物含量則沒有顯著影響。此部份之結果顯示CAPE在誘導HL-60細胞凋亡的過程中伴隨細胞內粒線體功能變化，因而可能因此導致GSH含量減少及過氧化氫之清除，亦可推知細胞內GSH含量在CAPE誘導HL-60細胞凋亡之過程中扮演重要角色。進一步研究CAPE誘導HL-60細胞凋亡之相關機轉，發現細胞經CAPE (6 ug/mL)處理後，依序發生下列變化：活化caspase-3 (4小時後)，抑制Bcl-2表現(6小時後)及增強Bax表現(16小時後)。
腫瘤細胞內GSH含量高是其對放射線治療具有抵抗力的重要因素，由於本研究證實CAPE具有降低細胞內GSH含量之作用，因此在以下的研究中，利用體外細胞模式及活體試驗探討CAPE是否對源自Fischer 344 大鼠的9L腦瘤及源自BALB/c小鼠的CT26大腸腺癌細胞株具有放射致敏作用。體外細胞模式結果顯示：在處理後20分鐘內CAPE即達9L細胞內最高藥物濃度，隨後即因而明顯降低9L及CT26細胞內GSH含量，但對正常骨髓細胞內之GSH含量則無明顯影響。CAPE 處理後， CT26細胞內的glutathione peroxidase (GPx) 活性增加，glutathione reductase活性減少，至於glutathione S-transferase及gamma-glutamyl transpeptidase活性則無明顯變化；進一步分析GPx各亞型的mRNA表現，發現在GPx的1-4亞型中，只有GPx —1亞型的 mRNA表現在CAPE處理後增加，其餘則無變化。 CAPE濃度在0 至 4 ug/mL範圍內對此二種腫瘤細胞並無明顯毒殺作用，以此範圍濃度CAPE預先處理9L及CT26細胞1小時後，再給予放射線(2 — 8 Gy)可明顯增強放射線之殺傷力達1.5到2.2倍，但若在CAPE處理前先給予NAC，則CAPE放射致敏作用會被減弱。利用cDNA微矩陣分析技術(cDNA microarray)以呈現CT26細胞經CAPE (2 ug/mL) 處理與否的基因表現，發現有明顯的基因表現差異，主要包含與GSH代謝、DNA修復、轉錄、訊息傳導及細胞黏附等相關之基因變化。
在探討CAPE對放射致敏作用的活體試驗中，首先將CT26腫瘤細胞植入BALB/c小鼠皮下，待腫瘤形成後，將不同濃度CAPE (0 — 20 mg/kg)經尾靜脈注射後，在不同時間(0 — 3 小時)犧牲動物後切下腫瘤測量腫瘤細胞內GSH含量，推測得知最適合放射致敏作用之給藥條件為：10 mg/kg 處理2小時。利用此投予條件並配合上述細胞植入模式，待腫瘤形成後將動物隨機分組，結合治療組的動物在單次10 Gy放射線治療前2小時，先自尾靜脈注射CAPE (10 mg/kg)，結果顯示結合治療組於放射線治療後可抑制腫瘤體積達54.1%，相較於單獨放射線處理組的腫瘤體積抑制程度(33.8%)，呈現明顯差異；結合治療並可延長小鼠平均存活時間及減少自發性肺臟轉移率。明顯有效之活體放射致敏結果亦見於分次(fractionated)給予放射治療以抑制腫瘤增長的同一動物模式中。在毒性評估方面，CAPE (10 mg/kg)注射對小鼠體重、肝、腎功能及骨髓造血功能並無明顯影響。
由本研究可知，CAPE能誘導人類白血病HL-60細胞凋亡，其間伴隨發生下列現象：粒線體功能變化、GSH含量驟減、H2O2含量劇減、caspase-3活化、Bcl-2表現減少及Bax表現增加。CAPE亦能大量降低大鼠9L腦瘤及小鼠CT26大腸癌細胞內的GSH含量，因而增加其對放射線之敏感度，此現象可能因為CAPE增強GPx-1 mRNA表現、增加GPx活性及減少GR活性所致。在適當給藥條件下，注射CAPE後再給予放射線治療可增加放射線對腫瘤體積抑制率，延長小鼠存活時間並減少自發性肺臟轉移率，同時對活體肝、腎及骨髓造血功能並無明顯影響。CAPE之放射致敏作用可能與GSH代謝及DNA修復等相關基因之調控有關。綜言之，CAPE為一有效的腫瘤凋亡誘導劑，且可運用於放射致敏而開發成為癌症輔助治療藥物，在癌症治療上具有潛力，且可能是安全的。

Caffeic acid phenethyl ester (CAPE), an active component of propolis, has many biological activities and pharmacological functions including anti-oxidation and tumor cell cytotoxicity. In this study, the type of cell death in human leukemic HL-60 cells after CAPE treatment and the relationship between CAPE-induced redox change and cell apoptosis have been elucidated. The mechanism of CAPE-induced radiosensitization in tumor cells and tumor-bearing animals were also studied. CAPE entered into HL-60 cells very rapidly and reduced cell viability in a concentration- and time-dependent manner. CAPE treatment (6 ug/mL) resulted in marked growth inhibition up to 70.3 +/- 4.0% at day 2. The inhibition of cell viability by CAPE was partially blocked by pretreatment with N-acetyl-L-cycteine (NAC). CAPE induced characteristic DNA fragmentation and morphological changes typical of cell apoptosis. Estimation of the apoptotic percentage showed a time-dependent increase after CAPE (6 ug/mL) treatment (up to 66.7 +/- 2.0% at 72 hour). CAPE induced a significant decrease in mitochondrial transmembrane potential to about half of the untreated level after 6 hours. A rapid depletion of intracellular glutathione (GSH) down to 41.7 +/- 6.0% was also observed after 1 hour of CAPE treatment. Pretreatment of HL-60 cells with NAC reversed the GSH depletion and partially rescued cells from CAPE-induced apoptosis. With regard to intracellular reactive oxygen species, CAPE caused a fast and profound scavenging of hydrogen peroxide (H2O2) (19% of that in untreated cells after a 2-hour treatment) but not of superoxide anion. These results suggest that apoptosis induced by CAPE is associated with mitochondrial dysfunction, GSH depletion and selective scavenging of H2O2 in human leukemic HL-60 cells. To investigate the detailed mechanism of CAPE-induced apoptosis in human leukemic HL-60 cells, several potential target molecules involved with cell apoptosis were assessed. Treatment with CAPE rapidly activated caspase-3 in 4 hours, down-regulated Bcl-2 expression in 6 hours, and up-regulated Bax expression in 16 hours.
Since CAPE can deprive intracellular GSH, an important intracellular molecule involved in radiation resistance, it is reasonable to explore the radiosensitizing potential of CAPE by using rat 9L glioma cells and mouse CT26 colorectal adenocarcinoma cells as in vitro and/or in vivo models. Results indicated that CAPE entered 9L cells very quickly and effectively depleted intracellular GSH within 30 min in 9L and CT26 cells, but not in bone marrow cells. The effects in activities of GSH metabolism enzymes, after CAPE treatment for 1 hour, were as it follows: increased in glutathione peroxidase and decreased in glutathione reductase without affecting glutathione S-transferase and gamma-glutamyl transpeptidase activities. In four various subtypes of GPx, only GPx-1 mRNA expression, as demonstrated by RT-PCR, was increased after CAPE treatment. The estimated nontoxic doses (> 90% survival) of CAPE ranged from 0 to 4 ug/mL. Pretreatment with this dose range of CAPE for 1 hour significantly enhanced cell killing by radiation with the sensitizer enhancement ratios (SER) ranging from 1.5 to 2.2 for 37% survival. The SER values of CAPE for 9L and CT26 cells increased with incremental doses of CAPE. Pretreatment of cells with NAC reversed the GSH depletion activity and partially blocked the radiosensitizing effect of CAPE. The cDNA microarray technique was used to explore the differential gene expression profiles after CAPE treatment. The up- and down-regulated genes identified were those involved in GSH metabolism, DNA repair, transcription, signal transduction, and cell adhesion.
In BALB/c mice implanted with CT26 tumor cells subcutaneously, pretreatment with CAPE [10 mg/kg via tail vein injection at 2 hours before radiation (10 Gy)] resulted in greater inhibition of tumor growth in comparison with radiation alone (54.1 vs 33.8% growth inhibition compared with controls on day 13). Moreover, pretreatment with CAPE prolonged the survival time and reduced the spontaneous pulmonary metastasis rate of tumor-bearing mice. Radiosensitization by CAPE pretreatment was also evident in tumor-bearing mice receiving fractionated radiation. CAPE treatment depleted intracellular GSH of tumor in vivo with an optimal dose of 10 mg/kg i.v. at 2 hours. Pretreatment with CAPE neither affected body weight nor produced liver, kidney or hematopoietic toxicity.
In brief, this study suggests that CAPE is a potent apoptosis-inducing agent whose action is accompanied by activation of caspase-3, down-regulation of Bcl-2 and up-regulation of Bax in human leukemic HL-60 cells. CAPE also depletes GSH in rat glioma 9L and mouse CT26 colorectal adenocarcinoma cells whose effect may be due to up-regulation of glutathione peroxidase-1 mRNA expression, activation of glutathione peroxidase and inhibition of glutathione reductase activity. Pretreatment with CAPE sensitizes 9L and CT26 cells to ionizing radiation. CAPE augments the inhibitory effect of ionizing radiation on implanted CT26 tumor, prolongs the survival, and reduces the pulmonary metastasis in BALA/c mice. There is no significant toxicity of CAPE on liver, kidney and bone marrow. This study concludes that CAPE is an effective apoptosis inducer and radiation sensitizer. It has potential in the adjunctive therapy of colorectal adenocarcinoma and malignant glioma with safety.

ABBREVIATIONS 3
摘 要 6
ABSTRACT 10
1. INTRODUCTION 14
1.1 Apoptosis 15
1.2 Reduction-Oxidation (Redox) State 19
1.3 Correlation between Redox State and Apoptosis 22
1.4 Propolis 24
1.5 Caffeic Acid Phenethyl Ester (CAPE) 26
1.6 Radiation Resistance of Tumor Cells and Radiation Sensitization 29
1.7 Rationale, Experimental Models, and Approaches of This Study 31
2. MATERIALS AND METHODS 32
2.1 Equipment 33
2.2 Materials 34
2.3 Methods 36
2.3.1 Cell Lines 36
2.3.2 CAPE, NAC and TPA Solution Preparation 38
2.3.3 Determination of Intracellular CAPE Concentration by HPLC 38
2.3.4 Cell Growth Kinetics 40
2.3.5 Micrographs 41
2.3.6 DNA Extraction and Gel Electrophoresis 42
2.3.7 Quantitation of Apoptosis by Flow Cytometer 42
2.3.8 Western Blotting 43
2.3.9 Analysis of Mitochondrial Transmembrane Potential 44
2.3.10 Fluorocytometric Analysis of GSH, H2O2 and Superoxide 45
2.3.11 Colorimetric Assay for Intracellular GSH Level 46
2.3.12 Assay for Cytosolic Glutathione Peroxidase (c-GPx) Activity 47
2.3.13 Assay for Cellular Glutathione Reductase (GR) Activity 47
2.3.14 Assay for gamma-Glutamyl Transpeptidase (gamma-GT) Activity 48
2.3.15 Assay for Glutathione S-Transferase (GST) Activity 48
2.3.16 Reverse Transcription and Polymerase Chain Reaction (RT-PCR) for GPx mRNA expression 49
2.3.17 CAPE Treatment and Radiation Delivery in Vitro 51
2.3.18 Tumor Cell Colony Assay 51
2.3.19 Syngenic Tumor Implantation Model 52
2.3.20 CAPE Administration and Intracellular GSH Levels in Vivo 52
2.3.21 CAPE Administration and Radiation Delivery in Vivo 53
2.3.22 Evaluation for Survival of Mice and Spontaneous Pulmonary Metastasis 54
2.3.23 Tumor Volume and Mouse Weight Measurements 54
2.3.24 Measurement of Plasma Levels of Alanine Aminotransferase (ALT) and Creatinine 54
2.3.25 Colony Forming Unit—Granulocytes/Macrophages (CFU-GM) Assay 55
2.3.26 cDNA Microarray Assay and Analysis 55
2.3.27 Statistics 56
3. RESULTS 58
3.1 The Time-Dependent CAPE Uptake by HL-60 And 9L Cells 58
3.2 Inhibition of Proliferation in HL-60 Cells by CAPE 58
3.3 Morphology of CAPE-treated HL-60 Cells Undergoing Apoptosis 59
3.4 Effect of CAPE to Induce Apoptosis 59
3.5 Expression of Bcl-2 and Bax, and Activity of Caspase-3 60
3.6 Reduction of CAPE-induced Growth Inhibition and Apoptosis by NAC 60
3.7 Mitochondrial Transmembrane Potential 61
3.8 Intracellular GSH Level 61
3.9 Intracellular ROS Level 62
3.10 Growth Kinetics in CAPE-treated 9L and CT26 Cells 62
3.11 Sensitization of 9L and CT26 Cells to Radiation by CAPE 62
3.12 Intracellular GSH Level in Vitro 63
3.13 Role of GSH in CAPE-Induced Radiosensitization 63
3.14 Colorimetric Aassay for Intracellular GSH Level in CT26 Cells and Normal Bone Marrow Cells after CAPE Treatment 63
3.15 Cytosolic GPx Activity in CT26 cells 64
3.16 Cellular GR Activity in CT26 cells 64
3.17 Cellular gamma-GT Activity in CT26 cells 65
3.18 Cellular GST Activity in CT26 cells 65
3.19 GPx mRNA Expression in CT26 Cells 65
3.20 Intracellular GSH Levels in Vivo 65
3.21 Radiosensitizing Effect of CAPE on Mice Bearing CT26 Colorectal Cancer Cells 65
3.22 CAPE and Ionizing Radiation on Survival of Mice Bearing CT26 Colorectal Cancer Cells 66
3.23 Spontaneous Pulmonary Metastasis in Mice Bearing CT26 Colorectal Cancer Cells 67
3.24 Body Weight, and Renal and Hepatic Functions in Tumor-Bearing Mice 67
3.25 CAPE Effects on Bone Marrow Cells 67
3.26 Differential Gene Expression Profiles after CAPE Treatment 68
4. DISCUSSION 70
4.1 Effective Dose of CAPE 70
4.2 Onset Time Course of CAPE and Apoptotic Machinery 71
4.3 CAPE and ROS 73
4.4 CAPE and GSH 75
4.5 Differential Effects of CAPE 77
4.6 CAPE in Radiosensitization 79
4.7 CAPE and Tumor Metastasis 81
4.8 Differential Gene Expression Profiles after CAPE Treatment 83
5. CONCLUSION 86
REFERENCES 87
Table 1-7…………………………………………………………………..……113-119
Figure 1-27………………………………………………………….……….…120-146
Scheme 1-8……………………………………………………………………..147-154
Appendix 1……………………………………………………………………..155-157
Reprints and manuscript

Abe, J., Okuda, M., Huang, Q., Yoshizumi, M., and Berk, B. C. (2000). Reactive oxygen species activate p90 ribosomal S6 kinase via Fyn and Ras. Journal of Biological Chemistry 275, 1739-1748.
Adler, V., Yin, Z., Tew, K. D., and Ronai, Z. (1999). Role of redox potential and reactive oxygen species in stress signaling. Oncogene 18, 6104-6111.
Alexander, E., III and Loeffler, J. S. (1998). Radiosurgery for primary malignant brain tumors. Seminars in Surgical Oncology 14, 43-52.
Aliev, B. A. (1968). Local use of the bee product Propolis in otorhinolaryngologic practice. Vestnik Otorinolaringologii 30, 105.
Arthur, J. R. (2000). The glutathione peroxidases. Cellular & Molecular Life Sciences 57, 1825-1835.
Badie, B., Drazan, K. E., Kramar, M. H., Shaked, A., and Black, K. L. (1995). Adenovirus-mediated p53 gene delivery inhibits 9L glioma growth in rats. Neurological Research 17, 209-216.
Banskota, A. H., Nagaoka, T., Sumioka, L. Y., Tezuka, Y., Awale, S., Midorikawa, K., Matsushige, K., and Kadota, S. (2002). Antiproliferative activity of the Netherlands propolis and its active principles in cancer cell lines. Journal of Ethnopharmacology 80, 67-73.
Benda, P., Someda, K., Messer, J., and Sweet, W. H. (1971). Morphological and immunochemical studies of rat glial tumors and clonal strains propagated in culture. Journal of Neurosurgery 34, 310-323.
Bhimani, R. S., Troll, W., Grunberger, D., and Frenkel, K. (1993). Inhibition of oxidative stress in HeLa cells by chemopreventive agents. Cancer Research 53, 4528-4533.
Biswal, S. S., Datta, K., Shaw, S. D., Feng, X., Robertson, J. D., and Kehrer, J. P. (2000). Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis in lipoxygenase-deficient FL5.12 cells. Toxicological Sciences 53, 77-83.
Boyd, J. M., Gallo, G. J., Elangovan, B., Houghton, A. B., Malstrom, S., Avery, B. J., Ebb, R. G., Subramanian, T., Chittenden, T., and Lutz, R. J. (1995). Bik, a novel death-inducing protein shares a distinct sequence motif with Bcl-2 family proteins and interacts with viral and cellular survival-promoting proteins. Oncogene 11, 1921-1928.
Brattain, M. G., Strobel-Stevens, J., Fine, D., Webb, M., and Sarrif, A. M. (1980). Establishment of mouse colonic carcinoma cell lines with different metastatic properties. Cancer Research 40, 2142-2146.
Brigelius-Flohe, R. (1999). Tissue-specific functions of individual glutathione peroxidases. Free Radical Biology & Medicine 27, 951-965.
Campling, B. G., Pym, J., Galbraith, P. R., and Cole, S. P. (1988). Use of the MTT assay for rapid determination of chemosensitivity of human leukemic blast cells. Leukemia Research 12, 823-831.
Cheng, K. C., Cahill, D. S., Kasai, H., Nishimura, S., and Loeb, L. A. (1992). 8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G-T and A-C substitutions. Journal of Biological Chemistry 267, 166-172.
Chiao, C., Carothers, A. M., Grunberger, D., Solomon, G., Preston, G. A., and Barrett, J. C. (1995). Apoptosis and altered redox state induced by caffeic acid phenethyl ester (CAPE) in transformed rat fibroblast cells. Cancer Research 55, 3576-3583.
Chiou, T. J. and Tzeng, W. F. (2000). The roles of glutathione and antioxidant enzymes in menadione-induced oxidative stress. Toxicology 154, 75-84.
Chittenden, T., Flemington, C., Houghton, A. B., Ebb, R. G., Gallo, G. J., Elangovan, B., Chinnadurai, G., and Lutz, R. J. (1995a). A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO Journal 14, 5589-5596.
Chittenden, T., Harrington, E. A., O''Connor, R., Flemington, C., Lutz, R. J., Evan, G. I., and Guild, B. C. (1995b). Induction of apoptosis by the Bcl-2 homologue Bak. Nature 20;374, 733-736.
Coates, A. and Tripp, E. (1995). Comparison of two fluorochromes for flow cytometric assay of cellular glutathione content in human malignant melanoma. Melanoma Research 5, 107-111.
Coffey, R. N., Watson, R. W., Hegarty, N. J., O''Neill, A., Gibbons, N., Brady, H. R., and Fitzpatrick, J. M. (2000). Thiol-mediated apoptosis in prostate carcinoma cells. Cancer 88, 2092-2104.
Cohen, G. M. (1997). Caspases: the executioners of apoptosis. Biochemical Journal 326, 1-16.
Cooper, J. S., Guo, M. D., Herskovic, A., Macdonald, J. S., Martenson, J. A., Jr., Al Sarraf, M., Byhardt, R., Russell, A. H., Beitler, J. J., Spencer, S., Asbell, S. O., Graham, M. V., and Leichman, L. L. (1999). Chemoradiotherapy of locally advanced esophageal cancer: long-term follow-up of a prospective randomized trial (RTOG 85-01). Radiation Therapy Oncology Group. JAMA 281, 1623-1627.
Cotter, T. G., Glynn, J. M., Echeverri, F., and Green, D. R. (1992). The induction of apoptosis by chemotherapeutic agents occurs in all phases of the cell cycle. Anticancer Research 12, 773-779.
Crompton, M. (1999). The mitochondrial permeability transition pore and its role in cell death. Biochemical Journal 341, 233-249.
Dou, Q. P., An, B., and Will, P. L. (1995). Induction of a retinoblastoma phosphatase activity by anticancer drugs accompanies p53-independent G1 arrest and apoptosis. Proceedings of the National Academy of Sciences of the United States of America 92, 9019-9023.
Fesen, M. R., Pommier, Y., Leteurtre, F., Hiroguchi, S., Yung, J., and Kohn, K. W. (1994). Inhibition of HIV-1 integrase by flavones, caffeic acid phenethyl ester (CAPE) and related compounds. Biochemical Pharmacology 48, 595-608.
Fisher, B., Wolmark, N., Rockette, H., Redmond, C., Deutsch, M., Wickerham, D. L., Fisher, E. R., Caplan, R., Jones, J., and Lerner, H. (1988). Postoperative adjuvant chemotherapy or radiation therapy for rectal cancer: results from NSABP protocol R-01. Journal of the National Cancer Institute 80, 21-29.
Frenkel, K. (1992). Carcinogen-mediated oxidant formation and oxidative DNA damage. Pharmacology & Therapeutics 53, 127-166.
Frenkel, K., Wei, H., Bhimani, R., Ye, J., Zadunaisky, J. A., Huang, M. T., Ferraro, T., Conney, A. H., and Grunberger, D. (1993). Inhibition of tumor promoter-mediated processes in mouse skin and bovine lens by caffeic acid phenethyl ester. Cancer Research 53, 1255-1261.
Gajate, C., Santos-Beneit, A. M., Macho, A., Lazaro, M., Hernandez-De Rojas, A., Modolell, M., Munoz, E., and Mollinedo, F. (2000). Involvement of mitochondria and caspase-3 in ET-18-OCH(3)-induced apoptosis of human leukemic cells. International Journal of Cancer 86, 208-218.
Garufi, C., Brienza, S., Pugliese, P., Aschelter, A. M., Bensmaine, Bertheault-Cvitkovic, F., Nistico, C., Giunta, S., Caterino, M., Giannarelli, D., Cosimelli, M., Levi, F., and Terzoli, E. (2000). Overcoming resistance to chronomodulated 5-fluorouracil and folinic acid by the addition of chronomodulated oxaliplatin in advanced colorectal cancer patients. Anti-Cancer Drugs 11, 495-501.
Gibson, D. D., Hawrylko, J., and McCay, P. B. (1985). GSH-dependent inhibition of lipid peroxidation: properties of a potent cytosolic system which protects cell membranes. Lipids 20, 704-711.
Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E. M., Efros, L., Caldwell, M., Estevez, V., and Nakanishi, K. (1988). Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia 44, 230-232.
Hegazi, A. G., Abd El Hady, F. K., and Abd Allah, F. A. (2000). Chemical composition and antimicrobial activity of European propolis. Zeitschrift fur Naturforschung Section C, Journal of Biosciences 55, 70-75.
Henning, G. T., Schild, S. E., Stafford, S. L., Donohue, J. H., Burch, P. A., Haddock, M. G., and Gunderson, L. L. (2000). Results of irradiation or chemoirradiation for primary unresectable, locally recurrent, or grossly incomplete resection of gastric adenocarcinoma. International Journal of Radiation Oncology, Biology, Physics. 46, 109-118.
Herrlich, P. and Bohmer, F. D. (2000). Redox regulation of signal transduction in mammalian cells. Biochemical Pharmacology 59, 35-41.
Hirpara, J. L., Seyed, M. A., Loh, K. W., Dong, H., Kini, R. M., and Pervaiz, S. (2000). Induction of mitochondrial permeability transition and cytochrome C release in the absence of caspase activation is insufficient for effective apoptosis in human leukemia cells. Blood 95, 1773-1780.
Holben, D. H. and Smith, A. M. (1999). The diverse role of selenium within selenoproteins: a review. Journal of the American Dietetic Association 99, 836-843.
Huang, Z. A., Yang, H., Chen, C., Zeng, Z., and Lu, S. C. (2000). Inducers of gamma-glutamylcysteine synthetase and their effects on glutathione synthetase expression. Biochimica et Biophysica Acta 1493, 48-55.
Jacobson, M. D. and Raff, M. C. (1995). Programmed cell death and Bcl-2 protection in very low oxygen. Nature 374, 814-816.
Jaiswal, A. K., Venugopal, R., Mucha, J., Carothers, A. M., and Grunberger, D. (1997). Caffeic acid phenethyl ester stimulates human antioxidant response element-mediated expression of the NAD(P)H:quinone oxidoreductase (NQO1) gene. Cancer Research 57, 440-446.
Janssen-Heininger, Y. M., Macara, I., and Mossman, B. T. (1999). Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-kappaB: requirement of Ras/mitogen-activated protein kinases in the activation of NF-kappaB by oxidants. American Journal of Respiratory Cell & Molecular Biology 20, 942-952.
Kaplowitz, N., Spina, C., Graham, M., and Kuhlenkamp, J. (1978). Glutathione S-transferase in human lymphoid cell lines and fractionated peripheral leucocytes. Biochemical Journal 169, 465-470.
Kerr, J. F., Wyllie, A. H., and Currie, A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26, 239-257.
Khachaturov, A. A. and Gudkov, A. I. (1969). Propolis therapy of certain dermatoses and burns in the far north. Vestnik Dermatologii i Venerologii 43, 63-65.
Kimoto, T., Aga, M., Hino, K., Koya-Miyata, S., Yamamoto, Y., Micallef, M. J., Hanaya, T., Arai, S., Ikeda, M., and Kurimoto, M. (2001). Apoptosis of human leukemia cells induced by Artepillin C, an active ingredient of Brazilian propolis. Anticancer Research 21, 221-228.
Klann, E., Roberson, E. D., Knapp, L. T., and Sweatt, J. D. (1998). A role for superoxide in protein kinase C activation and induction of long-term potentiation. Journal of Biological Chemistry 20;273, 4516-4522.
Koltuksuz, U., Ozen, S., Uz, E., Aydinc, M., Karaman, A., Gultek, A., Akyol, O., Gursoy, M. H., and Aydin, E. (1999). Caffeic acid phenethyl ester prevents intestinal reperfusion injury in rats. Journal of Pediatric Surgery 34, 1458-1462.
Kretz-Remy, C., Mehlen, P., Mirault, M. E., and Arrigo, A. P. (1996). Inhibition of I kappa B-alpha phosphorylation and degradation and subsequent NF-kappa B activation by glutathione peroxidase overexpression. Journal of Cell Biology 133, 1083-1093.
Kroemer, G., Zamzami, N., and Susin, S. A. (1997). Mitochondrial control of apoptosis. Immunology Today 18, 44-51.
Lafrenie, R., Shaughnessy, S. G., and Orr, F. W. (1992). Cancer cell interactions with injured or activated endothelium. Cancer & Metastasis Reviews 11, 377-388.
Lee, Y. J., Liao, P. H., Chen, W. K., and Yang, C. Y. (2000). Preferential cytotoxicity of caffeic acid phenethyl ester analogues on oral cancer cells. Cancer Letters 153, 51-56.
Lepekhin, V. N. and Leonova, T. A. (1970). Antimicrobial properties of propolis. Stomatologiia 49, 16-19.
Liebmann, J., Cook, J. A., Fisher, J., Teague, D., and Mitchell, J. B. (1994). In vitro studies of Taxol as a radiation sensitizer in human tumor cells. Journal of the National Cancer Institute 86, 441-446.
Lin, S. C., Chung, C. Y., Chiang, C. L., and Hsu, S. H. (1999). The influence of propolis ethanol extract on liver microsomal enzymes and glutathione after chronic alcohol administration. American Journal of Chinese Medicine 27, 83-93.
Lin, S. C., Lin, Y. H., Chen, C. F., Chung, C. Y., and Hsu, S. H. (1997). The hepatoprotective and therapeutic effects of propolis ethanol extract on chronic alcohol-induced liver injuries. American Journal of Chinese Medicine 25, 325-332.
Lopez, J. B. and Buttery, J. E. (1977). Colorimetric gamma-glutamyltransferase assay: non-inhibition by p-nitroaniline. Clinica Chimica Acta 77, 91-93.
Luo, J., Soh, J. W., Xing, W. Q., Mao, Y., Matsuno, T., and Weinstein, I. B. (2001). PM-3, a benzo-gamma-pyran derivative isolated from propolis, inhibits growth of MCF-7 human breast cancer cells. Anticancer Research 21, 1665-1671.
Marcucci, M. C., Ferreres, F., Garcia-Viguera, C., Bankova, V. S., De Castro, S. L., Dantas, A. P., Valente, P. H., and Paulino, N. (2001). Phenolic compounds from Brazilian propolis with pharmacological activities. Journal of Ethnopharmacology 74, 105-112.
McGowan, A. J., Fernandes, R. S., Verhaegen, S., and Cotter, T. G. (1994). Zinc inhibits UV radiation-induced apoptosis but fails to prevent subsequent cell death. International Journal of Radiation Biology 66, 343-349.
Medical, R. (2001). Randomized trial of procarbazine, lomustine, and vincristine in the adjuvant treatment of high-grade astrocytoma: a Medical Research Council trial. Journal of Clinical Oncology 19, 509-518.
Meister, A. (1994). Glutathione, ascorbate, and cellular protection. Cancer Research 54, Suppl-1975s.
Merino, N., Gonzalez, R., Gonzalez, A., and Remirez, D. (1996). Histopathological evaluation on the effect of red propolis on liver damage induced by CCl4 in rats. Archives of Medical Research 27, 285-289.
Michaluart, P., Masferrer, J. L., Carothers, A. M., Subbaramaiah, K., Zweifel, B. S., Koboldt, C., Mestre, J. R., Grunberger, D., Sacks, P. G., Tanabe, T., and Dannenberg, A. J. (1999). Inhibitory effects of caffeic acid phenethyl ester on the activity and expression of cyclooxygenase-2 in human oral epithelial cells and in a rat model of inflammation. Cancer Research 59, 2347-2352.
Midgley, R. and Kerr, D. (1999). Colorectal cancer. Lancet 353 , 391-399.
Mirkovic, N., Voehringer, D. W., Story, M. D., McConkey, D. J., McDonnell, T. J., and Meyn, R. E. (1997). Resistance to radiation-induced apoptosis in Bcl-2-expressing cells is reversed by depleting cellular thiols. Oncogene 15, 1461-1470.
Mirzoeva, O. K. and Calder, P. C. (1996). The effect of propolis and its components on eicosanoid production during the inflammatory response. Prostaglandins Leukotrienes & Essential.Fatty Acids 55, 441-449.
Mitchell, S. E., Mendenhall, W. M., Zlotecki, R. A., and Carroll, R. R. (2001). Squamous cell carcinoma of the anal canal. International Journal of Radiation Oncology, Biology, Physics. 49, 1007-1013.
Munoz, O., Pena, R. C., Ureta, E., Montenegro, G., Caldwell, C., and Timmermann, B. N. (2001). Phenolic compounds of propolis from Central Chilean matorral. Zeitschrift fur Naturforschung Section C, Journal of Biosciences 56, 273-277.
Muschel, R. J., Bernhard, E. J., Garza, L., McKenna, W. G., and Koch, C. J. (1995). Induction of apoptosis at different oxygen tensions: evidence that oxygen radicals do not mediate apoptotic signaling. Cancer Research 55, 995-998.
Nakajima, Y., Miyamoto, T., Tanabe, M., Watanabe, I., and Terasima, T. (1979). Enhancement of mammalian cell killing by 5-fluorouracil in combination with X-rays. Cancer Research 39, 3763-3767.
Natarajan, K., Singh, S., Burke, T. R., Jr., Grunberger, D., and Aggarwal, B. B. (1996). Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B. Proceedings of the National Academy of Sciences of the United States of America 20;93, 9090-9095.
Newkirk, K., Heffern, J., Sloman-Moll, E., Sessions, R. B., Rasmussen, A. A., Andrews, P. A., and Cullen, K. J. (1997). Glutathione content but not gamma glutamyl cysteine synthetase mRNA expression predicts cisplatin resistance in head and neck cancer cell lines. Cancer Chemotherapy & Pharmacology 40, 75-80.
Nomura, M., Kaji, A., Ma, W., Miyamoto, K., and Dong, Z. (2001). Suppression of cell transformation and induction of apoptosis by caffeic acid phenethyl ester. Molecular Carcinogenesis 31, 83-89.
O''Connell, M. J., Martenson, J. A., Wieand, H. S., Krook, J. E., Macdonald, J. S., Haller, D. G., Mayer, R. J., Gunderson, L. L., and Rich, T. A. (1994). Improving adjuvant therapy for rectal cancer by combining protracted-infusion fluorouracil with radiation therapy after curative surgery. New England Journal of Medicine 331, 502-507.
Oberley, L. W. and Buettner, G. R. (1979). Role of superoxide dismutase in cancer: a review. Cancer Research 39, 1141-1149.
Oda, T., Iwaoka, J., Komatsu, N., and Muramatsu, T. (1999). Involvement of N-acetylcysteine-sensitive pathways in ricin-induced apoptotic cell death in U937 cells. Bioscience Biotechnology & Biochemistry 63, 341-348.
Orr, F. W., Adamson, I. Y., Warner, D., Leroyer, V., Werner, L., Shaughnessy, S., and Young, L. (1988). The effects of oxygen radical--mediated pulmonary endothelial damage on cancer metastasis. Molecular & Cellular Biochemistry 84, 189-198.
Ota, C., Unterkircher, C., Fantinato, V., and Shimizu, M. T. (2001). Antifungal activity of propolis on different species of Candida. Mycoses 44, 375-378.
Ovchinnikov, V. I. (1965). Propolis in the therapy of some dermatoses. Vestnik Dermatologii i Venerologii 39, 69-70.
Paglia, D. E. and Valentine, W. N. (1967). Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. Journal of Laboratory & Clinical Medicine 70, 158-169.
Palazzo, J. P., Kafka, N. J., Grasso, L., Chakrani, F., Hanau, C., Cuesta, K. H., and Mercer, W. E. (1997). The role of p53, p21WAF1/C1PI, and bcl-2 in radioresistant colorectal carcinoma. Human Pathology 28, 1189-1195.
Pan, M. H., Chang, W. L., Lin-Shiau, S. Y., Ho, C. T., and Lin, J. K. (2001). Induction of apoptosis by garcinol and curcumin through cytochrome c release and activation of caspases in human leukemia HL-60 cells. Journal of Agricultural & Food Chemistry 49, 1464-1474.
Pan, M. H., Liang, Y. C., Lin-Shiau, S. Y., Zhu, N. Q., Ho, C. T., and Lin, J. K. (2000). Induction of apoptosis by the oolong tea polyphenol theasinensin A through cytochrome c release and activation of caspase-9 and caspase-3 in human U937 cells. Journal of Agricultural & Food Chemistry 48, 6337-6346.
Pascual, C., Gonzalez, R., and Torricella, R. G. (1994). Scavenging action of propolis extract against oxygen radicals. Journal of Ethnopharmacology 41, 9-13.
Pinto, R. E. and Bartley, W. (1968). Changes in glutathione reductase and glutathione peroxidase activities in rat liver related to age and sex. Biochemical Journal 109, 34.
Prados, M. D., Wara, W. M., Sneed, P. K., McDermott, M., Chang, S. M., Rabbitt, J., Page, M., Malec, M., Davis, R. L., Gutin, P. H., Lamborn, K., Wilson, C. B., Phillips, T. L., and Larson, D. A. (2001). Phase III trial of accelerated hyperfractionation with or without difluromethylornithine (DFMO) versus standard fractionated radiotherapy with or without DFMO for newly diagnosed patients with glioblastoma multiforme. International Journal of Radiation Oncology, Biology, Physics 49, 71-77.
Rahman, I. (2000). Regulation of nuclear factor-kappa B, activator protein-1, and glutathione levels by tumor necrosis factor-alpha and dexamethasone in alveolar epithelial cells. Biochemical Pharmacology 60, 1041-1049.
Rahman, I. and MacNee, W. (2000). Regulation of redox glutathione levels and gene transcription in lung inflammation: therapeutic approaches. Free Radical Biology & Medicine 28, 1405-1420.
Rose, P. G., Bundy, B. N., Watkins, E. B., Thigpen, J. T., Deppe, G., Maiman, M. A., Clarke-Pearson, D. L., and Insalaco, S. (1999). Concurrent cisplatin-based radiotherapy and chemotherapy for locally advanced cervical cancer. New England Journal of Medicine 340 , 1144-1153.
Rottenberg, H. and Wu, S. (1998). Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochimica et Biophysica Acta 1404, 393-404.
Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tobiume, K., Sawada, Y., Kawabata, M., Miyazono, K., and Ichijo, H. (1998). Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO Journal 17, 2596-2606.
Schulze-Osthoff, K., Los, M., and Baeuerle, P. A. (1995). Redox signalling by transcription factors NF-kappa B and AP-1 in lymphocytes. Biochemical Pharmacology 50, 735-741.
Sen, C. K. and Packer, L. (1996). Antioxidant and redox regulation of gene transcription. FASEB Journal 10, 709-720.
Skarlatos, J., Kosma, L., Koukourakis, M., Zambatis, C., Kapsoritakis, A., Mouder, N., and Yannakakis, D. (1996). Hypofractionated radiotherapy with concurrent 5-fluorouracil radiosensitisation for recurrent or locally advanced colorectal cancer. A phase II study. International Journal of Colorectal Disease 11, 206-210.
Smaaland, R., Svardal, A. M., Lote, K., Ueland, M., and Laerum, O. D. (1991). Glutathione content in human bone marrow and circadian stage relation to DNA synthesis. Journal of the National Cancer Institute 83, 1092-1098.
Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150, 76-85.
St Clair, D. K. and Holland, J. C. (1991). Complementary DNA encoding human colon cancer manganese superoxide dismutase and the expression of its gene in human cells. Cancer Research 51, 939-943.
Strand, S., Hofmann, W. J., Hug, H., Muller, M., Otto, G., Strand, D., Mariani, S. M., Stremmel, W., Krammer, P. H., and Galle, P. R. (1996). Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells--a mechanism of immune evasion? Nature Medicine 2, 1361-1366.
Sud''ina, G. F., Mirzoeva, O. K., Pushkareva, M. A., Korshunova, G. A., Sumbatyan, N. V., and Varfolomeev, S. D. (1993). Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Letters 329, 21-24.
Takahashi, A., Alnemri, E. S., Lazebnik, Y. A., Fernandes-Alnemri, T., Litwack, G., Moir, R. D., Goldman, R. D., Poirier, G. G., Kaufmann, S. H., and Earnshaw, W. C. (1996). Cleavage of lamin A by Mch2 alpha but not CPP32: multiple interleukin 1 beta-converting enzyme-related proteases with distinct substrate recognition properties are active in apoptosis. Proceedings of the National Academy of Sciences of the United States of America 93, 8395-8400.
Tewari, M., Quan, L. T., O''Rourke, K., Desnoyers, S., Zeng, Z., Beidler, D. R., Poirier, G. G., Salvesen, G. S., and Dixit, V. M. (1995). Yama/CPP32 beta, a mammalian homolog of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly(ADP-ribose) polymerase. Cell 81, 801-809.
Thomas, C. R., Jr., Weiden, P. L., Traverso, L. W., and Thompson, T. (1997). Concomitant intraarterial cisplatin, intravenous 5-flourouracil, and split-course radiation therapy for locally advanced unresectable pancreatic adenocarcinoma: a phase II study of the Puget Sound Oncology Consortium (PSOC-703). American Journal of Clinical Oncology 20, 161-165.
Thornberry, N. A. and Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 1312-1316.
Tsai, T. H., Chen, Y. F., Chen, I. F., and Chen, C. F. (1999). Measurement of unbound caffeic acid in rat blood by on-line microdialysis coupled with liquid chromatography and its application to pharmacokinetic study. Journal of Chromatography B, Biomedical Sciences & Applications 729, 119-125.
Vaux, D. L., Weissman, I. L., and Kim, S. K. (1992). Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2. Science 258, 1955-1957.
Wang, I. K., Lin-Shiau, S. Y., and Lin, J. K. (1999). Induction of apoptosis by apigenin and related flavonoids through cytochrome c release and activation of caspase-9 and caspase-3 in leukaemia HL-60 cells. European Journal of Cancer 35, 1517-1525.
Wang, J. F., Jerrells, T. R., and Spitzer, J. J. (1996b). Decreased production of reactive oxygen intermediates is an early event during in vitro apoptosis of rat thymocytes. Free Radical Biology & Medicine 20, 533-542.
Wang, S. Y., Chen, L. Y., Tsai, T. F., Su, T. S., Choo, K. B., and Ho, C. K. (1996a). Constitutive production of colony-stimulating factors by human hepatoma cell lines: possible correlation with cell differentiation. Experimental Hematology 24, 437-444.
Wang, S. Y., Hsu, M. L., Hsu, H. C., Tzeng, C. H., Lee, S. S., Shiao, M. S., and Ho, C. K. (1997). The anti-tumor effect of Ganoderma lucidum is mediated by cytokines released from activated macrophages and T lymphocytes. International Journal of Cancer 70, 699-705.
Xu, Y., Krishnan, A., Wan, X. S., Majima, H., Yeh, C. C., Ludewig, G., Kasarskis, E. J., and St Clair, D. K. (1999). Mutations in the promoter reveal a cause for the reduced expression of the human manganese superoxide dismutase gene in cancer cells. Oncogene 18, 93-102.
Yang, E. B., Tang, W. Y., Zhang, K., Cheng, L. Y., and Mack, P. O. (1997). Norcantharidin inhibits growth of human HepG2 cell-transplanted tumor in nude mice and prolongs host survival. Cancer Letters 117, 93-98.
Yi, X., Ding, L., Jin, Y., Ni, C., and Wang, W. (1994). The toxic effects, GSH depletion and radiosensitivity by BSO on retinoblastoma. International Journal of Radiation Oncology, Biology, Physics 29, 393-396.
Zhang, X., Neamati, N., Lee, Y. K., Orr, A., Brown, R. D., Whitaker, N., Pommier, Y., and Burke, T. R., Jr. (2001). Arylisothiocyanate-containing esters of caffeic acid designed as affinity ligands for HIV-1 integrase. Bioorganic & Medicinal Chemistry 9, 1649-1657.
Zhong, W., Oberley, L. W., Oberley, T. D., Yan, T., Domann, F. E., and St Clair, D. K. (1996). Inhibition of cell growth and sensitization to oxidative damage by overexpression of manganese superoxide dismutase in rat glioma cells. Cell Growth & Differentiation 7, 1175-1186.