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Immunomodulatory activity of cucurbitacin E isolated from Ecballium elaterium
Fitoterapia 76 (2005) 439 – 441 www.elsevier.com/locate/fitote
Short report
Immunomodulatory activity of cucurbitacin E isolated from Ecballium elaterium E. Attarda,*, M.P. Brincatb, A. Cuschieric a Institute of Agriculture, University of Malta, Malta Department of Obstetrics and Gynaecology, Faculty of Medicine and Surgery, University of Malta, Malta c Department of Anatomy, Faculty of Medicine and Surgery, University of Malta, Malta
b
Received 20 October 2004; received in revised form 24 February 2005; accepted 24 February 2005 Available online 23 May 2005
Abstract The immunomodulatory effect of cucurbitacin E, extracted from Ecballium elaterium, was tested on peripheral human lymphocytes. These lymphocytes were co-cultured with cancer cells and an interesting lymphocyte-mediated cytotoxicity was observed. D 2005 Elsevier B.V. All rights reserved. Keywords: Ecballium elaterium; Immunomodulatory activity; Cucurbitacin E
1. Plant material Ecballium elaterium L., A. Richard (Cucurbitaceae) [1], juice of mature fruit collected in December 2001 from the university campus of Msida, was identified at the Institute of Agriculture, University of Malta, where a voucher specimen is deposited.
2. Uses in traditional medicine E. elaterium has been used to treat constipation, rheumatism, and jaundice, and as an emetic [2]. * Corresponding author. Fax: +356 21346519. E-mail address: [email protected] (E. Attard). 0367-326X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2005.02.007
440
E. Attard et al. / Fitoterapia 76 (2005) 439–441
3. Previously isolated constituents Previously isolated constituents were carbohydrates, gum, leucoanthocyanins, tannins, cucurbitacins, and peptides (E. elaterium protease inhibitors) [3].
4. Tested materials Tested materials were cucurbitacin E (CuE) [4] and phytohemagglutinin (PHA), purchased from Gibco (UK).
5. Studied activity The cytotoxicity of CuE and/or PHA-stimulated human peripheral lymphocytes against human cancer cell lines was tested using the microtitration colorimetric method of WST-1, tetrazolium salt (Boehringer Mannheim, Germany) [5]. Compounds were dissolved in dimethylsulphoxide (DMSO) and diluted in the medium at a predetermined concentration, as indicated in previous experiments [6], prior to application. After 48 h of incubation of the lymphocytes with the four treatment combinations, the cells were washed and cocultured with the cancer cell lines for another 48 h, after which cell proliferation was determined.
6. Used cell lines Two human cancer cell lines, PC-3 (prostate cancer) and ZR-75-1 (breast cancer), were used and obtained from the European Tissue Culture Collection (Porton Down, Salisbury, UK). The PC-3 and ZR-75-1 cell lines were maintained in Ham’s F12 with 7% foetal calf serum (FCS) and Roswell Park Memorial Institute (RPMI) 1640 with 10% FCS, respectively, with 100 U/ml penicillin and 100 Ag/ml streptomycin. Cultures were held in 75-cm2 culture flasks at 37 8C, 5% CO2, and 90% relative humidity, changing the media at least twice a week. The peripheral human lymphocytes were obtained from fresh human Table 1 Cytotoxicity of treated and untreated lymphocytes on PC-3 and ZR-75-1 cell linesa Cell lines
PC-3 ZR-75-1 a b c d e
Lymphocytes treated with CuEb
PHAc
PHA/CuEd
Controle
80.777 F 13.985 86.617 F 3.598
84.373 F 10.740 88.657 F 3.387
66.900 F 14.533 45.377 F 10.740
85.653 F 14.771 90.317 F 16.896
Percentage cytotoxicity (%) is the mean of at least three determinations FS.D. Final concentration was 20 AM. Final concentration was 1%. Final concentration: CuE 20 AM and PHA 1%. Untreated cells.
E. Attard et al. / Fitoterapia 76 (2005) 439–441
441
blood from a healthy male volunteer. These were isolated using Histopaque-1077 (Sigma, USA). Cells were washed and maintained in RPMI 1640 medium with 10% FCS, antibiotics and PHA, PHA/CuE, and CuE treatments. The untreated cells were considered as negative control.
7. Results The results are reported in Table 1.
8. Conclusions The cytotoxic enhancement of the lymphocytes against both cell lines was less pronounced in control cultures than the treated ones. In fact, the best result was obtained for the PHA/CuE combination-treated lymphocytes. The effects of PHA and CuE were additive in the case of PC-3 cells but synergistic in the case of ZR-75-1 cells. This confirms the fact that PC-3 cells are resistant to lymphocyte-mediated cytotoxicity [6]. The PHA/CuE combination on ZR-75-1 cell line indicated that the cytotoxic T-cell subset was activated and effectively led to ZR-75-1 cell death. This goes in accordance with another experiment [7], where activated lymphocytes released IL-6 [8], an interleukin that has considerable activity against ZR-75-1 cells in vitro. From previous studies [9], the direct effect of CuE on PC-3 cells was more pronounced than on the ZR-75-1 cells. As a result of this investigation, the potential cell-mediated effects of CuE on the cancer cell lines are demonstrated.
References [1] Costich DE. Plant Genet Resour Newsl 1997;112:98. [2] Lanfranco G. Hxejjex Medicinali u Ohrajn Fil-Gzejjer Maltin. Pubblikazzjoni Media Centre, 1993. [3] Jodral MM, Martin JJ, Agil AM, Navarro Moll MC, Cabo Cires MP. Boletin da Sociedade Bioteriana 1990;63:213. [4] Attard E. CGC Reports 2002;25:71. [5] Trauth BC, Keesey J. Guide to Cell Proliferation and Apoptosis Methods. Mannheim7 Boehringer; 1997. [6] Borsellino N, Bonavida B, Ciliberto G, Toniatti C, Travali S, D’Alessandro N. Cancer 1999;85:134. [7] Asgeirsson KS, Olafsdottir K, Jonasson JG, Ogmundsdottir HM. Cytokine 1998;10:720. [8] Feldmann M. Cell cooperation in the antibody. In: Roitt I, Brostoff J, Male D, editors. Immunology. Mandarin Offset (HK) Ltd.; 1993. p. 7.7–7.14. [9] Attard E, Cuschieri A. J Nat Rem 2004;4:137.
Short report
Immunomodulatory activity of cucurbitacin E isolated from Ecballium elaterium E. Attarda,*, M.P. Brincatb, A. Cuschieric a Institute of Agriculture, University of Malta, Malta Department of Obstetrics and Gynaecology, Faculty of Medicine and Surgery, University of Malta, Malta c Department of Anatomy, Faculty of Medicine and Surgery, University of Malta, Malta
b
Received 20 October 2004; received in revised form 24 February 2005; accepted 24 February 2005 Available online 23 May 2005
Abstract The immunomodulatory effect of cucurbitacin E, extracted from Ecballium elaterium, was tested on peripheral human lymphocytes. These lymphocytes were co-cultured with cancer cells and an interesting lymphocyte-mediated cytotoxicity was observed. D 2005 Elsevier B.V. All rights reserved. Keywords: Ecballium elaterium; Immunomodulatory activity; Cucurbitacin E
1. Plant material Ecballium elaterium L., A. Richard (Cucurbitaceae) [1], juice of mature fruit collected in December 2001 from the university campus of Msida, was identified at the Institute of Agriculture, University of Malta, where a voucher specimen is deposited.
2. Uses in traditional medicine E. elaterium has been used to treat constipation, rheumatism, and jaundice, and as an emetic [2]. * Corresponding author. Fax: +356 21346519. E-mail address: [email protected] (E. Attard). 0367-326X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2005.02.007
440
E. Attard et al. / Fitoterapia 76 (2005) 439–441
3. Previously isolated constituents Previously isolated constituents were carbohydrates, gum, leucoanthocyanins, tannins, cucurbitacins, and peptides (E. elaterium protease inhibitors) [3].
4. Tested materials Tested materials were cucurbitacin E (CuE) [4] and phytohemagglutinin (PHA), purchased from Gibco (UK).
5. Studied activity The cytotoxicity of CuE and/or PHA-stimulated human peripheral lymphocytes against human cancer cell lines was tested using the microtitration colorimetric method of WST-1, tetrazolium salt (Boehringer Mannheim, Germany) [5]. Compounds were dissolved in dimethylsulphoxide (DMSO) and diluted in the medium at a predetermined concentration, as indicated in previous experiments [6], prior to application. After 48 h of incubation of the lymphocytes with the four treatment combinations, the cells were washed and cocultured with the cancer cell lines for another 48 h, after which cell proliferation was determined.
6. Used cell lines Two human cancer cell lines, PC-3 (prostate cancer) and ZR-75-1 (breast cancer), were used and obtained from the European Tissue Culture Collection (Porton Down, Salisbury, UK). The PC-3 and ZR-75-1 cell lines were maintained in Ham’s F12 with 7% foetal calf serum (FCS) and Roswell Park Memorial Institute (RPMI) 1640 with 10% FCS, respectively, with 100 U/ml penicillin and 100 Ag/ml streptomycin. Cultures were held in 75-cm2 culture flasks at 37 8C, 5% CO2, and 90% relative humidity, changing the media at least twice a week. The peripheral human lymphocytes were obtained from fresh human Table 1 Cytotoxicity of treated and untreated lymphocytes on PC-3 and ZR-75-1 cell linesa Cell lines
PC-3 ZR-75-1 a b c d e
Lymphocytes treated with CuEb
PHAc
PHA/CuEd
Controle
80.777 F 13.985 86.617 F 3.598
84.373 F 10.740 88.657 F 3.387
66.900 F 14.533 45.377 F 10.740
85.653 F 14.771 90.317 F 16.896
Percentage cytotoxicity (%) is the mean of at least three determinations FS.D. Final concentration was 20 AM. Final concentration was 1%. Final concentration: CuE 20 AM and PHA 1%. Untreated cells.
E. Attard et al. / Fitoterapia 76 (2005) 439–441
441
blood from a healthy male volunteer. These were isolated using Histopaque-1077 (Sigma, USA). Cells were washed and maintained in RPMI 1640 medium with 10% FCS, antibiotics and PHA, PHA/CuE, and CuE treatments. The untreated cells were considered as negative control.
7. Results The results are reported in Table 1.
8. Conclusions The cytotoxic enhancement of the lymphocytes against both cell lines was less pronounced in control cultures than the treated ones. In fact, the best result was obtained for the PHA/CuE combination-treated lymphocytes. The effects of PHA and CuE were additive in the case of PC-3 cells but synergistic in the case of ZR-75-1 cells. This confirms the fact that PC-3 cells are resistant to lymphocyte-mediated cytotoxicity [6]. The PHA/CuE combination on ZR-75-1 cell line indicated that the cytotoxic T-cell subset was activated and effectively led to ZR-75-1 cell death. This goes in accordance with another experiment [7], where activated lymphocytes released IL-6 [8], an interleukin that has considerable activity against ZR-75-1 cells in vitro. From previous studies [9], the direct effect of CuE on PC-3 cells was more pronounced than on the ZR-75-1 cells. As a result of this investigation, the potential cell-mediated effects of CuE on the cancer cell lines are demonstrated.
References [1] Costich DE. Plant Genet Resour Newsl 1997;112:98. [2] Lanfranco G. Hxejjex Medicinali u Ohrajn Fil-Gzejjer Maltin. Pubblikazzjoni Media Centre, 1993. [3] Jodral MM, Martin JJ, Agil AM, Navarro Moll MC, Cabo Cires MP. Boletin da Sociedade Bioteriana 1990;63:213. [4] Attard E. CGC Reports 2002;25:71. [5] Trauth BC, Keesey J. Guide to Cell Proliferation and Apoptosis Methods. Mannheim7 Boehringer; 1997. [6] Borsellino N, Bonavida B, Ciliberto G, Toniatti C, Travali S, D’Alessandro N. Cancer 1999;85:134. [7] Asgeirsson KS, Olafsdottir K, Jonasson JG, Ogmundsdottir HM. Cytokine 1998;10:720. [8] Feldmann M. Cell cooperation in the antibody. In: Roitt I, Brostoff J, Male D, editors. Immunology. Mandarin Offset (HK) Ltd.; 1993. p. 7.7–7.14. [9] Attard E, Cuschieri A. J Nat Rem 2004;4:137.