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Anticancer activity of Hemsleya amabilis extract
Life Sciences 71 (2002) 2161 – 2170 www.elsevier.com/locate/lifescie
Anticancer activity of Hemsleya amabilis extract Jin Wu a,*, Yaojiong Wu b, Burton B. Yang b a
Room 108, South Wing, Patrick Manson Building, The University of Hong Kong, 7 Sassoon Road, Pokfulam, Hong Kong, China b Sunnybrook and Women’s College Health Sciences Centre, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Received 28 March 2002; accepted 14 May 2002
Abstract Hemsleya amabilis extract is derived from the medicinal herb Hemsleya amabilis, which has long been used to treat cancer and many other conditions. The underlying mechanism is not clear. To investigate Hemsleya amabili’s anticancer activity, we have treated different types of cancer cells including human astrocytoma U87 cells, breast cancer cells MDA-MB-231and Jurkat cells with Hemsleya amabilis extract. This agent significantly inhibited tumor cell growth and colony formation at various concentrations. When astrocytoma cells were seeded in the presence of Hemsleya amabilis extract at very low concentrations, cell spreading was greatly inhibited. Hemsleya amabilis extract also promoted tumor cell death in all the tested cell lines, but with varied sensitivities. Apoptotic assays with Annexin V staining demonstrated that Hemsleya amabilis extract induced astrocytoma cell apoptosis at different concentrations. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Hemsleya; Anticancer herbs; Cell growth; Apoptosis; Colony formation
Introduction Many medical herbs have long been used as anticancer agents [1–5]. Emerging evidence has demonstrated that many natural products isolated from plant sources possess antitumor properties [6– 14]. Apoptosis, a specific form of programmed cell death, plays an important role in development, growth regulation, diseases of the heart, immune system, neurodegenerative disorders, as well as tumor *
Corresponding author. Tel.: +852-2819-9133; fax: +852-2816-5704. E-mail address: [email protected] (J. Wu). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 0 1 3 - 1
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development and therapy [15–18]. Apoptotic programs are present in latent form in virtually all cell types throughout the body [19]. Once triggered by a variety of physiologic signals, this program initiates in a precisely choreographed manner. Cell membranes are disrupted, the cytoplasmic and nuclear skeletons are broken down, and the DNA is fragmented. Eventually the cell corpses are engulfed by a nearby cell in the tissue and disappear [20]. Apoptosis is an important physiological process in maintaining balanced tissue cell regeneration. Tumor growth is determined not only by the rate of cell proliferation, but also by the rate of cell death [21]. Mounting evidence [22,23], principally from studies in mouse models and cultured cells, as well as from descriptive analyses of biopsied tissues in human carcinogenesis, suggests that resistance toward apoptosis is a hallmark of most and perhaps all types of cancers. Inhibiting tumor cell growth and inducing tumor cell apoptosis are ideal therapeutic strategies. Among a variety of pro-apoptotic agents, plant-derived compounds are characterized by their multiple mechanisms and low side effects [1–4,6,24]. Hemsleya amabilis extract is a compound prepared from the plant named Hemsleya amabilis. Hemsleya amabilis has long been used in China to treat cancers as well as infectious diseases [25]. However, the mechanisms of its anti-cancer properties are barely understood. In this study, we found that Hemsleya amabilis extract significantly inhibited tumor cell growth, colony formation and induced tumor cell apoptosis.
Materials and methods Materials Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS) and trypsin/EDTA were from Invitrogen (Burlington, Ontario, Canada). Tissue culture plates were from Nunc Inc. U87 astrocytoma cell line, MDA-MB-231 human breast cancer cell line and Jurkat cell line were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in DMEM supplemented with 10% FBS at 37 jC in a humidified incubator containing 5% CO2. Annextin V apoptosis detection kit (sc-4252) was from Santa Cruz Biotechnology Inc. All chemicals were from Sigma (St. Louis, MO). Hemsleya amabilis extract is a compound derived by extraction from dry Hemsleya amabilis. Briefly, dry Hemsleya amabilis was cleaned and ground into small pieces, followed by incubation with 5 times (v/w) ethanol (95%) at room temperature for 2 h. The ethanol was recovered and the remaining pellet was subjected to the same procedure for one more extraction. Ethanol from the two extractions was pooled, and an ethyl acetate/methanol (1:1) mixture was added. The extract was powdered by vacuum drying. Apoptosis assay Aapoptosis was assayed by detecting the expression of annexin V on the membrane of apoptotic cells [26,27]. Annexin V-FITC conjugates bind to the negatively charged phosphatidylserine and allow for detection of early apoptotic cells, preceding DNA fragmentation and membrane disruption. Apoptotic cells stained with annexin V-FITC were quantified by flow cytometry (Becton Dickinson). The assay was performed following the instructions of the manufacturer. Briefly, 2 105 U87 astrocytoma cells were seeded on 6-well tissue culture plates in DMEM supplemented with 10% FBS in the presence and
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absence of Hemsleya amabilis extract at different concentrations as indicated in each figure and maintained at 37 jC in a humidified incubator containing 5% CO2 for different intervals. U87 cell cultures without Hemsleya amabilis extract treatment were used as controls. The cultured cells were detached by tripsinization and pelleted by centrifugation at 1400 rpm for 10 min. The cells were washed twice with cold PBS and resuspended in 1 assay buffer (provided by Santa Cruz) to obtain a cell density of 1 106 cells/ml. Annexin V-FITC (8 Al) was added to 400 Al of cell aliquot (2 105 cells) followed by an incubation at room temperature in the dark for 15 min. The cells were then washed with cold PBS and subjected to flow cytometry analysis immediately. Cell viability assay Cells were seeded on 6-well tissue culture plates at the density of 2 105 cell per well in DMEM supplemented with 10% FBS in the presence and absence (as controls) of Hemsleya amabilis extract at different concentrations as indicated. Culture medium was used to dilute Hemsleya amabilis extract. The cultures were maintained at 37 jC in a tissue incubator containing 5% CO2 for defined time periods. The cultured cells were then detached by trypsinization and stained with trypan blue. The dead cells and the total cells were counted. Viable cells (%) = [ (total cells dead cells) / total cells ] 100%. Cell number determination The effect of Hemsleya amabilis extract on tumor cell growth was measured by cell counting using a cytometer [28]. The cells were seeded at a density of 5 103 cells per well to 96-well tissue culture plates in triplets in DMEM supplemented with 10% FBS in the presence or absence of Hemsleya amabilis extract at concentrations diluted with culture medium. The cultures were maintained in an incubator at 37 jC for 72 h. The cells were harvested by trypsinizatioin and cell number was counted using a cytometor. The numbers of cells were analyzed statistically using t–test. Colony formation assay To understand how Hemsleya amabilis extract inhibits tumor growth, we performed a colony formation assay in a soft agarose gel. Briefly, 6-well tissue culture plates were coated with 1% agarose. U87 human astrocytoma cells and MDA-MB-231 breast cancer cells were mixed with 0.4% soft agarose in DMEM with 10% FBS supplementation at 37 jC. The cells were plated at a density of 103 cells per well in triplets in the absence or presence of Hemsleya amabilis extract at different concentrations. The cultures were incubated at 37 jC in a humidified incubator containing 5% CO2 for 2 weeks. Colonies z 50 Am in diameter were counted [29]. The number of colonies was analyzed statistically using t–test.
Results Hemsleya amabilis extract inhibits tumor cell growth The effect of Hemsleya amabilis extract on tumor cell growth was examined on U87 human astrocytoma cell line and MDA-MB-231 human breast cancer cell line. Hemsleya amabilis extract
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inhibited the growth of both cell lines in a similar fashion. Result from one typical experiment (U87 cells) is shown in Fig. 1. At low concentrations, the extracts inhibited the tumor cell growth. With increased concentrations, the tumor cell growth was completely blocked. Hemsleya amabilis extract inhibits colony formation Transformed cells have the property to form tumors in vivo and to form colonies in soft agarose gel. Tumor cell lines are transformed cells and are able to form colonies in soft agarose gel. We employed this system to test the effect of Hemsleya amabilis extract on colony formation. U87 human astrocytoma cell line and MDA-MB-231 breast cancer cell line were mixed with 0.4% soft agarose in the presence or absence of Hemsleya amabilis extract at various concentrations and plated on top of tissue culture plates coated with 0.6% agarose gel. Hemsleya amabilis extract significantly inhibited colony formations of U87 cells (Fig. 2A) and MDA-MB-231 cells (Fig. 2B) at all concentrations used ( V 12.5 Ag/ml). With increased concentrations, Hemsleya amabilis extract completely abolished the tumor cell colony formations (Fig. 2A and 2B). A typical experimental result in colony formation treated with or without Hemsleya amabilis extract is shown (Fig. 2C and 2D). This inhibitory effect of Hemsleya amabilis extract on colony formation was similar to its effect on cell growth (Fig. 1).
Fig. 1. Hemsleya amabilis extract inhibits tumor cell growth. Astrocytoma cell line U87 was seeded on 96-well tissue culture plates at a density of 5 103 cells/well in DMEM supplemented with 10% FBS. Hemsleya amabilis extract diluted with culture medium was added to the cultures at final concentrations of 0, 12.5, 25, and 50 Ag/ml, and the cultures were maintained at 37 jC for 72 h in a tissue culture incubator. The cells were harvested by trypsinization and cell number was counted using a cytometer. Cell number declined by the Hemsleya amabilis treatment (n = 3, *, p V 0.01).
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Fig. 2. Hemsleya amabilis extract inhibited colony formation. U87 cells (A) and MDA-MB-231 cells (B) were mixed in 0.4% low melting agarose in DMEM supplemented with 10% FBS in the presence of Hemsleya amabilis extract at concentrations of 0, 12.5, 25, and 50 Ag/ml. After 2 weeks incubation, colonies ( z 50 Am in diameter) were counted. Each data point represents the mean F S.E. of three individual experiments. Colony formation was also photographed. A typical result of MDA-MB-231 colony formation treated with (C) or without (D) Hemsleya amabilis extract (50 Ag/ml) is shown.
Hemsleya amabilis extract induces tumor cell death The decrease in cell number treated with Hemsleya amabilis extract suggested cell death in the cell cultures. To confirm this, we conducted cell viability assay stained with trypan blue. Cultures of the U87 astrocytoma cell line, MDA-MB-231 breast cancer cell line and Jurkat cells were treated with Hemsleya amabilis extract at various concentrations for 24 to 72 h. The results indicated that Hemsleya amabilis extract had cytotoxicity on all the tumor cells tested. Twenty-four hours after the addition of Hemsleya amabilis extract, all tumor cells started dying (data not shown). After 72 h incubation, cell death was obvious even at low concentration treatments. However, the sensitivities varied. U87 cells were the most sensitive to Hemsleya amabilis treatment: over 50% of cells died when treated with 25 Ag/ml of Hemsleya amabilis extract (Fig. 3A). At this concentration, only 40% of MDA-MB-231 cells died, and a concentration of 50 Ag/ml was required to obtain 50% cell death (Fig. 3B). Jurkat cells were the most insensitive group to Hemsleya amabilis treatment. To obtain 50% cell death, a concentration of 150 Ag/ ml Hemsleya amabilis extract was required (Fig. 3C).
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Fig. 3. Hemsleya amabilis extract induces tumor cell death. U87 cells (A), MDA-MB-231 cells (B) and Jurkat cells (C) were seeded on 6-well tissue culture plates (2 105 cells/well) in DMEM supplemented with 10% FBS. Hemsleya amabilis extract was added to the cultures at concentrations of 0, 25, 50, 100, 150, 200, and 300 Ag/ml, followed by an incubation. The cultures were maintained at 37 jC for 72 h. The cells were harvested by trypsinization, stained with trypan blue and the viable cells were counted. Hemsleya amabilis extract induced tumor cell death (n = 3).
To understand how Hemsleya amabilis extract induced cell death, we examined cell morphology affected by this compound. U87 astrocytoma cells were treated with or without Hemsleya amabilis extract. We demonstrated that cell spreading and elongation of U87 astrocytoma cells was severely affected by Hemsleya amabilis treatment at a concentration of 25 Ag/ml for 24 hours (Fig. 4B) compared with the untreated control (Fig. 4A). Treatment with 25 mg/ml of Hemsleya amabilis extract for 48 hours reduced the number of cells attached to plates (Fig. 4C). These results suggest that Hemsleya amabilis
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Fig. 4. Hemsleya amabilis extract inhibited tumor cell spreading. U87 cells were seeded on 6-well tissue culture plates (2 105 cells/well) DMEM supplemented with 10% FBS in the absence and presence of Hemsleya amabilis extract and incubated for 24 h at 37 jC. Without Hemsleya amabilis treatment, U87 cells spread well on the plates with extended processes (A). In the presence of Hemsleya amabilis extract (25 Ag/ml) for 24 hours (B) and 48 hours (C), cell spreading was greatly inhibited: most of the cells rounded up.
extract inhibited the interactions of cells with extracellular matrix. On the other hand, Jurkat cells, which could grow anchorage-independently, died at significantly higher concentrations compared with U87 and MDA-MB-231 cells (data not shown).
Fig. 5. Hemsleya amabilis extract promotes apoptosis of astrocytoma cells. U87 cells were seeded on 6-well tissue culture plates (2 105 cells/well) in DMEM supplemented with 10% FBS in the presence of Hemsleya amabilis extract at the final concentrations of 0, 25, and 50 Ag/ml. The cultures were maintained at 37 jC for 72 h. The cells were harvested by trypsinization, stained with annexin V-FITC, and analyzed on flow cytometry. The percentages indicate the proportion of apoptotic cells in the whole population.
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To investigate if the cells died necrotically and/or apoptoticly, we examined cell apoptosis after Hemsleya amabilis extract treatment. U87 cells were treated with or without Hemsleya amabilis extract. Apoptotic cells were detected using an annexin V-FITC apoptosis marker and quantified by flow cytometry. Massive cell apoptosis was found in Hemsleya amabilis extract treated cells (Fig. 5). Comparing with cell viabilities (Fig. 4), higher proportions of cells had undergone apoptosis. This was due to the high sensitivity of annexin V in apoptosis detection. Both the cell death and apoptosis were in a dosage-dependent manner (Fig. 3 and Fig. 5).
Discussion Hemsleya amabilis is an herb widely growing in many provinces in China. Hemsleya amabilis has been used in the treatment of many infectious illnesses. In recent years it has been used clinically to treat cancers and has been demonstrated an ability to greatly inhibit tumor development and growth with limited side effects [25]. However, the mechanism underlying its anti-tumor effect is not clear. In this study, we investigated the effect of Hemsleya amabilis extract on cell activities. We demonstrated that Hemsleya amabilis extract had significant effects on inhibiting tumor cell growth, colony formation and inducing tumor cell apoptosis. Inhibiting tumor growth has been a continuous effort in cancer treatment. A reduction in cell growth and an induction in cell death are two major means to inhibit tumor growth. In this study, we demonstrated that, at low concentrations, Hemsleya amabilis extract caused significant inhibition of growth in U87 astrocytoma cell line, MDA-MB-231 breast cancer cell line and Jurkat cells. These cells were derived from different tissue sources. The inhibitory effect of Hemsleya amabilis extract on cell growth implies that this compound may have a general function in anti-tumor cell growth. This is not unexpected, since cancer cells have developed the capacity of increased proliferation through a variety of growth signal pathways. This includes elevated external growth factors, increased intracellular matrix signal via integrin [30], and Ras protein mutation-derived constitutive mitogenic signals [31], resulting in growing neoplasm, that causes destruction and atrophy of the surrounding tissue and adjacent organs. In a specific tumor, one pathway may play a more important role than the others. Hemsleya amabilis extract may act on more than one pathway. Nevertheless, we did observe different sensitivities of tumor cells to the growth inhibitory effect of treatments of Hemsleya amabilis extract. Jurkat cells, for example, which were poorly anchorage-dependent in growth and survival, were less sensitive to the treatments as compared with U87 astracytoma cell line and MDA-MB-231 breast cancer cells, which were anchoragedependent in growth. This is coincident with the findings that Hemsleya amabilis extract inhibited U87 cell spreading. These results suggest that Hemsleya amabilis extract may interfere with the interaction of tumor cells with ECM, or signals of the interaction. To develop a tumor, cancer cells have to form a nude in vivo as the first step. We investigated the effect of Hemsleya amabilis extract on colony formation in vitro. Cancer cells are able to form colonies in soft agarose gel, and this was confirmed in the controls, in which no Hemsleya amabilis extract was added to the soft agarose gel cultures. The addition of Hemsleya amabilis extract inhibited colony formation completely. This result may explain the anti-cancer effect of Hemsleya amabilis extract in clinical application. Normal organ development is controlled by a balance between cell proliferation and apoptosis [19]. In cancer, the balance between proliferation and programmed cell death is disturbed. There is strong
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evidence that tumour growth is a result of uncontrolled proliferation and reduced apoptosis [32,33]. Cancer cells have an acquired capability to evade apoptosis through a variety of ways [32,33]. Inducing tumor cell apoptosis is an ideal way to kill cancer cells. In this study, when different human tumor cells were treated with Hemsleya amabilis extract, they underwent apoptotic death. However, different sensitivities were observed among different tumor cells. In a similar fashion, adherent astrocytoma and breast cancer cells are more sensitive to Hemsleya amabilis extract, compared with anchorageindependent Jurkat cells. Apoptosis is modulated by anti-apoptotic and pro-apoptotic effectors, which involve a large number of proteins [19,32,33]. Hemsleya amabilis extract-induced tumor cell apoptosis may involve an array of mechanisms. However, the difference in sensitivity to Hemsleya amabilisinduced apoptosis between the adherent and non-anchorage-dependent cells suggest that Hemsleya amabilis extract may inhibit ECM-derived cell survival signals, which play an important anti-apoptotic role [34,35]. Furthermore, the fact that the extracts induce non-anchorage dependent Jurkat cell apoptosis at increased concentrations suggests that other mechanisms are involved. Our results suggest that the anticancer effect of Hemsleya amabilis is a combination of its effects in inhibiting tumor cell growth and inducing tumor cell apoptosis. It thus has a great advantage over other types of treatments such as chemotherapy and more recently, hormonal treatments. Most of the chemotherapy treatments fail due to drug resistance. As an herb extract, Hemsleya amabilis contains a variety of compounds that may act on different pathways of tumor cell growth and survival. The molecular mechanisms underlying these effects await further investigation. Acknowledgements This work is supported by the University of Hong Kong. References [1] Pezzuto JM. Plant-derived anticancer agents. Biochemical Pharmacology 1997;53(2):121 – 33. [2] DiPaola RS, Zhang H, Lambert GH, Meeker R, Licitra E, Rafi MM, Zhu BT, Spaulding H, Goodin S, Toledano MB, Hait WN, Gallo MA. Clinical and biologic activity of an estrogenic herbal combination (PC-SPES) in prostate cancer. New England Journal of Medicine 1998;339(12):785 – 91. [3] Tiwari RK, Geliebter J, Garikapaty VP, Yedavelli SP, Chen S, Mittelman A. Anti-tumor effects of PC-SPES, an herbal formulation in prostate cancer. International Journal of Oncology 1999;14(4):713 – 9. [4] Darzynkiewicz Z, Traganos F, Wu JM, Chen S. Chinese herbal mixture PC SPES in treatment of prostate cancer (review). International Journal of Oncology 2000;17(4):729 – 36. [5] Duong Van Huyen JP, Sooryanarayana, Delignat S, Bloch MF, Kazatchkine MD, Kaveri SV. Variable sensitivity of lymphoblastoid cells to apoptosis induced by Viscum album Qu FrF, a therapeutic preparation of mistletoe lectin. Chemotherapy 2001;47(5):366 – 76. [6] Sakamoto S, Kudo H, Kuwa K, Suzuki S, Kato T, Kawasaki T, Nakayama T, Kasahara N, Okamoto R. Anticancer effects of a Chinese herbal medicine, juzen-taiho-to, in combination with or without 5-fluorouracil derivative on DNA-synthesizing enzymes in 1,2-dimethylhydrazine induced colonic cancer in rats. American Journal of Chinese Medicine 1991;19 (3 – 4):233 – 41. [7] Prozesky EA, Meyer JJ, Louw AI. In vitro antiplasmodial activity and cytotoxicity of ethnobotanically selected South African plants. Journal of Ethnopharmacology 2001;76(3):239 – 45. [8] Kawada M, Ohno Y, Ri Y, Ikoma T, Yuugetu H, Asai T, Watanabe M, Yasuda N, Akao S, Takemura G, Minatoguchi S, Gotoh K, Fujiwara H, Fukuda K. Anti-tumor effect of gallic acid on LL-2 lung cancer cells transplanted in mice. Anticancer Drugs 2001;12(10):847 – 52.
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[9] Moalic S, Liagre B, Corbiere C, Bianchi A, Dauca M, Bordji K, Beneytout JL. A plant steroid, diosgenin, induces apoptosis, cell cycle arrest and COX activity in osteosarcoma cells. FEBS Letter 2001;506(3):225 – 30. [10] Park HI, Jeong MH, Lim YJ, Park BS, Kim GC, Lee YM, Kim HM, Yoo KS, Yoo YH. Szygium aromaticum (L.) Merr. Et Perry (Myrtaceae) flower bud induces apoptosis of p815 mastocytoma cell line. Life Sciences 2001;69 (5):553 – 66. [11] Gupta S, Hastak K, Ahmad N, Lewin JS, Mukhtar H. Inhibition of prostate carcinogenesis in TRAMP mice by oral infusion of green tea polyphenols. Proceedings of the National Academic Sciences U S A 2001;98(18):10350 – 5. [12] Luo J, Soh JW, Xing WQ, Mao Y, Matsuno T, Weinstein IB. PM-3, a benzo-gamma-pyran derivative isolated from propolis, inhibits growth of MCF-7 human breast cancer cells. Anticancer Research 2001;21(3B):1665 – 71. [13] Mujoo K, Haridas V, Hoffmann JJ, Wachter GA, Hutter LK, Lu Y, Blake ME, Jayatilake GS, Bailey D, Mills GB, Gutterman JU. Triterpenoid saponins from Acacia victoriae (Bentham) decrease tumor cell proliferation and induce apoptosis. Cancer Research 2001;61(14):5486 – 90. [14] Chang MC, Ho YS, Lee PH, Chan CP, Lee JJ, Hahn LJ, Wang YJ, Jeng JH. Areca nut extract and arecoline induced the cell cycle arrest but not apoptosis of cultured oral KB epithelial cells: association of glutathione, reactive oxygen species and mitochondrial membrane potential. Carcinogenesis 2001;22(9):1527 – 35. [15] Wyllie AH, Bellamy CO, Bubb VJ, Clarke AR, Corbet S, Curtis L, Harrison DJ, Hooper ML, Toft N, Webb S, Bird CC. Apoptosis and carcinogenesis. British Journal of Cancer 1999;80(Suppl 1):34 – 7. [16] Parton M, Dowsett M, Smith I. Studies of apoptosis in breast cancer. British Medical Journal 2001;322(7301):1528 – 32. [17] Lin JD. The role of apoptosis in autoimmune thyroid disorders and thyroid cancer. British Medical Journal 2001; 322(7301):1525 – 7. [18] Gibson RM. Does apoptosis have a role in neurodegeneration? British Medical Journal 2001;322(7301):1539 – 40. [19] Reed JC. Mechanisms of apoptosis. American Journal of Pathology 2000;157(5):1415 – 30. [20] Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. International Review of Cytology 1980;68: 251 – 306. [21] Reed JC. Dysregulation of apoptosis in cancer. Journal of Clinical Oncology 1999;17(9):2941 – 53. [22] Sarraf CE, Bowen ID. Proportions of mitotic and apoptotic cells in a range of untreated experimental tumours. Cell and Tissue Kinetics 1988;21(1):45 – 9. [23] Carson DA, Ribeiro JM. Apoptosis and disease. Lancet 1993;341(8855):1251 – 4. [24] Porterfield H. UsToo PC-SPES surveys: review of studies and update of previous survey results. Molecular Urology 2000;4(3):289 – 91 (discussion 293). [25] Niu CQ, He LY. [HPTLC separation and fluorodensitometric determination of isoquinoline alkaloids in Chelidonine majus L]. Yao Xue Xue Bao 1992;27(1):69 – 73. [26] Chao C, Saito S, Kang J, Anderson CW, Appella E, Xu Y. p53 transcriptional activity is essential for p53-dependent apoptosis following DNA damage. The EMBO Journal 2000;19(18):4967 – 75. [27] Vitale C, Romagnani C, Puccetti A, Olive D, Costello R, Chiossone L, Pitto A, Bacigalupo A, Moretta L, Mingari MC. Surface expression and function of p75/AIRM-1 or CD33 in acute myeloid leukemias: engagement of CD33 induces apoptosis of leukemic cells. Proceedings of the National Academic Sciences U S A 2001;98(10):5764 – 9. [28] Wu Y, Zhang Y, Cao L, Chen L, Lee V, Zheng PS, Kiani C, Adams ME, Ang LC, Paiwand F, Yang BB. Identification of the motif in versican G3 domain that plays a dominant-negative effect on astrocytoma cell proliferation through inhibiting versican secretion and binding. The Journal of Biological Chemistry 2001;276(17):14178 – 86. [29] Busse D, Doughty RS, Ramsey TT, Russell WE, Price JO, Flanagan WM, Shawver LK, Arteaga CL. Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity. The Journal of Biological Chemistry 2000;275(10):6987 – 95. [30] Lukashev ME, Werb Z. ECM signalling: orchestrating cell behaviour and misbehaviour. Trends in Cell Biology 1998;8(11):437 – 41. [31] Medema RH, Bos JL. The role of p21ras in receptor tyrosine kinase signaling. Critical Review of Oncology 1993; 4(6):615 – 61. [32] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100(1):57 – 70. [33] Evan GI, Vousden KH. Proliferation, cell cycle and apoptosis in cancer. Nature 2001;411(6835):342 – 8. [34] Ishizaki Y, Cheng L, Mudge AW, Raff MC. Programmed cell death by default in embryonic cells, fibroblasts, and cancer cells. Molecular Biology of the Cell 1995;6(11):1443 – 58. [35] Giancotti FG, Ruoslahti E. Integrin signaling. Science 1999;285(5430):1028 – 32.
Anticancer activity of Hemsleya amabilis extract Jin Wu a,*, Yaojiong Wu b, Burton B. Yang b a
Room 108, South Wing, Patrick Manson Building, The University of Hong Kong, 7 Sassoon Road, Pokfulam, Hong Kong, China b Sunnybrook and Women’s College Health Sciences Centre, and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Received 28 March 2002; accepted 14 May 2002
Abstract Hemsleya amabilis extract is derived from the medicinal herb Hemsleya amabilis, which has long been used to treat cancer and many other conditions. The underlying mechanism is not clear. To investigate Hemsleya amabili’s anticancer activity, we have treated different types of cancer cells including human astrocytoma U87 cells, breast cancer cells MDA-MB-231and Jurkat cells with Hemsleya amabilis extract. This agent significantly inhibited tumor cell growth and colony formation at various concentrations. When astrocytoma cells were seeded in the presence of Hemsleya amabilis extract at very low concentrations, cell spreading was greatly inhibited. Hemsleya amabilis extract also promoted tumor cell death in all the tested cell lines, but with varied sensitivities. Apoptotic assays with Annexin V staining demonstrated that Hemsleya amabilis extract induced astrocytoma cell apoptosis at different concentrations. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Hemsleya; Anticancer herbs; Cell growth; Apoptosis; Colony formation
Introduction Many medical herbs have long been used as anticancer agents [1–5]. Emerging evidence has demonstrated that many natural products isolated from plant sources possess antitumor properties [6– 14]. Apoptosis, a specific form of programmed cell death, plays an important role in development, growth regulation, diseases of the heart, immune system, neurodegenerative disorders, as well as tumor *
Corresponding author. Tel.: +852-2819-9133; fax: +852-2816-5704. E-mail address: [email protected] (J. Wu). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 0 1 3 - 1
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development and therapy [15–18]. Apoptotic programs are present in latent form in virtually all cell types throughout the body [19]. Once triggered by a variety of physiologic signals, this program initiates in a precisely choreographed manner. Cell membranes are disrupted, the cytoplasmic and nuclear skeletons are broken down, and the DNA is fragmented. Eventually the cell corpses are engulfed by a nearby cell in the tissue and disappear [20]. Apoptosis is an important physiological process in maintaining balanced tissue cell regeneration. Tumor growth is determined not only by the rate of cell proliferation, but also by the rate of cell death [21]. Mounting evidence [22,23], principally from studies in mouse models and cultured cells, as well as from descriptive analyses of biopsied tissues in human carcinogenesis, suggests that resistance toward apoptosis is a hallmark of most and perhaps all types of cancers. Inhibiting tumor cell growth and inducing tumor cell apoptosis are ideal therapeutic strategies. Among a variety of pro-apoptotic agents, plant-derived compounds are characterized by their multiple mechanisms and low side effects [1–4,6,24]. Hemsleya amabilis extract is a compound prepared from the plant named Hemsleya amabilis. Hemsleya amabilis has long been used in China to treat cancers as well as infectious diseases [25]. However, the mechanisms of its anti-cancer properties are barely understood. In this study, we found that Hemsleya amabilis extract significantly inhibited tumor cell growth, colony formation and induced tumor cell apoptosis.
Materials and methods Materials Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS) and trypsin/EDTA were from Invitrogen (Burlington, Ontario, Canada). Tissue culture plates were from Nunc Inc. U87 astrocytoma cell line, MDA-MB-231 human breast cancer cell line and Jurkat cell line were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in DMEM supplemented with 10% FBS at 37 jC in a humidified incubator containing 5% CO2. Annextin V apoptosis detection kit (sc-4252) was from Santa Cruz Biotechnology Inc. All chemicals were from Sigma (St. Louis, MO). Hemsleya amabilis extract is a compound derived by extraction from dry Hemsleya amabilis. Briefly, dry Hemsleya amabilis was cleaned and ground into small pieces, followed by incubation with 5 times (v/w) ethanol (95%) at room temperature for 2 h. The ethanol was recovered and the remaining pellet was subjected to the same procedure for one more extraction. Ethanol from the two extractions was pooled, and an ethyl acetate/methanol (1:1) mixture was added. The extract was powdered by vacuum drying. Apoptosis assay Aapoptosis was assayed by detecting the expression of annexin V on the membrane of apoptotic cells [26,27]. Annexin V-FITC conjugates bind to the negatively charged phosphatidylserine and allow for detection of early apoptotic cells, preceding DNA fragmentation and membrane disruption. Apoptotic cells stained with annexin V-FITC were quantified by flow cytometry (Becton Dickinson). The assay was performed following the instructions of the manufacturer. Briefly, 2 105 U87 astrocytoma cells were seeded on 6-well tissue culture plates in DMEM supplemented with 10% FBS in the presence and
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absence of Hemsleya amabilis extract at different concentrations as indicated in each figure and maintained at 37 jC in a humidified incubator containing 5% CO2 for different intervals. U87 cell cultures without Hemsleya amabilis extract treatment were used as controls. The cultured cells were detached by tripsinization and pelleted by centrifugation at 1400 rpm for 10 min. The cells were washed twice with cold PBS and resuspended in 1 assay buffer (provided by Santa Cruz) to obtain a cell density of 1 106 cells/ml. Annexin V-FITC (8 Al) was added to 400 Al of cell aliquot (2 105 cells) followed by an incubation at room temperature in the dark for 15 min. The cells were then washed with cold PBS and subjected to flow cytometry analysis immediately. Cell viability assay Cells were seeded on 6-well tissue culture plates at the density of 2 105 cell per well in DMEM supplemented with 10% FBS in the presence and absence (as controls) of Hemsleya amabilis extract at different concentrations as indicated. Culture medium was used to dilute Hemsleya amabilis extract. The cultures were maintained at 37 jC in a tissue incubator containing 5% CO2 for defined time periods. The cultured cells were then detached by trypsinization and stained with trypan blue. The dead cells and the total cells were counted. Viable cells (%) = [ (total cells dead cells) / total cells ] 100%. Cell number determination The effect of Hemsleya amabilis extract on tumor cell growth was measured by cell counting using a cytometer [28]. The cells were seeded at a density of 5 103 cells per well to 96-well tissue culture plates in triplets in DMEM supplemented with 10% FBS in the presence or absence of Hemsleya amabilis extract at concentrations diluted with culture medium. The cultures were maintained in an incubator at 37 jC for 72 h. The cells were harvested by trypsinizatioin and cell number was counted using a cytometor. The numbers of cells were analyzed statistically using t–test. Colony formation assay To understand how Hemsleya amabilis extract inhibits tumor growth, we performed a colony formation assay in a soft agarose gel. Briefly, 6-well tissue culture plates were coated with 1% agarose. U87 human astrocytoma cells and MDA-MB-231 breast cancer cells were mixed with 0.4% soft agarose in DMEM with 10% FBS supplementation at 37 jC. The cells were plated at a density of 103 cells per well in triplets in the absence or presence of Hemsleya amabilis extract at different concentrations. The cultures were incubated at 37 jC in a humidified incubator containing 5% CO2 for 2 weeks. Colonies z 50 Am in diameter were counted [29]. The number of colonies was analyzed statistically using t–test.
Results Hemsleya amabilis extract inhibits tumor cell growth The effect of Hemsleya amabilis extract on tumor cell growth was examined on U87 human astrocytoma cell line and MDA-MB-231 human breast cancer cell line. Hemsleya amabilis extract
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inhibited the growth of both cell lines in a similar fashion. Result from one typical experiment (U87 cells) is shown in Fig. 1. At low concentrations, the extracts inhibited the tumor cell growth. With increased concentrations, the tumor cell growth was completely blocked. Hemsleya amabilis extract inhibits colony formation Transformed cells have the property to form tumors in vivo and to form colonies in soft agarose gel. Tumor cell lines are transformed cells and are able to form colonies in soft agarose gel. We employed this system to test the effect of Hemsleya amabilis extract on colony formation. U87 human astrocytoma cell line and MDA-MB-231 breast cancer cell line were mixed with 0.4% soft agarose in the presence or absence of Hemsleya amabilis extract at various concentrations and plated on top of tissue culture plates coated with 0.6% agarose gel. Hemsleya amabilis extract significantly inhibited colony formations of U87 cells (Fig. 2A) and MDA-MB-231 cells (Fig. 2B) at all concentrations used ( V 12.5 Ag/ml). With increased concentrations, Hemsleya amabilis extract completely abolished the tumor cell colony formations (Fig. 2A and 2B). A typical experimental result in colony formation treated with or without Hemsleya amabilis extract is shown (Fig. 2C and 2D). This inhibitory effect of Hemsleya amabilis extract on colony formation was similar to its effect on cell growth (Fig. 1).
Fig. 1. Hemsleya amabilis extract inhibits tumor cell growth. Astrocytoma cell line U87 was seeded on 96-well tissue culture plates at a density of 5 103 cells/well in DMEM supplemented with 10% FBS. Hemsleya amabilis extract diluted with culture medium was added to the cultures at final concentrations of 0, 12.5, 25, and 50 Ag/ml, and the cultures were maintained at 37 jC for 72 h in a tissue culture incubator. The cells were harvested by trypsinization and cell number was counted using a cytometer. Cell number declined by the Hemsleya amabilis treatment (n = 3, *, p V 0.01).
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Fig. 2. Hemsleya amabilis extract inhibited colony formation. U87 cells (A) and MDA-MB-231 cells (B) were mixed in 0.4% low melting agarose in DMEM supplemented with 10% FBS in the presence of Hemsleya amabilis extract at concentrations of 0, 12.5, 25, and 50 Ag/ml. After 2 weeks incubation, colonies ( z 50 Am in diameter) were counted. Each data point represents the mean F S.E. of three individual experiments. Colony formation was also photographed. A typical result of MDA-MB-231 colony formation treated with (C) or without (D) Hemsleya amabilis extract (50 Ag/ml) is shown.
Hemsleya amabilis extract induces tumor cell death The decrease in cell number treated with Hemsleya amabilis extract suggested cell death in the cell cultures. To confirm this, we conducted cell viability assay stained with trypan blue. Cultures of the U87 astrocytoma cell line, MDA-MB-231 breast cancer cell line and Jurkat cells were treated with Hemsleya amabilis extract at various concentrations for 24 to 72 h. The results indicated that Hemsleya amabilis extract had cytotoxicity on all the tumor cells tested. Twenty-four hours after the addition of Hemsleya amabilis extract, all tumor cells started dying (data not shown). After 72 h incubation, cell death was obvious even at low concentration treatments. However, the sensitivities varied. U87 cells were the most sensitive to Hemsleya amabilis treatment: over 50% of cells died when treated with 25 Ag/ml of Hemsleya amabilis extract (Fig. 3A). At this concentration, only 40% of MDA-MB-231 cells died, and a concentration of 50 Ag/ml was required to obtain 50% cell death (Fig. 3B). Jurkat cells were the most insensitive group to Hemsleya amabilis treatment. To obtain 50% cell death, a concentration of 150 Ag/ ml Hemsleya amabilis extract was required (Fig. 3C).
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Fig. 3. Hemsleya amabilis extract induces tumor cell death. U87 cells (A), MDA-MB-231 cells (B) and Jurkat cells (C) were seeded on 6-well tissue culture plates (2 105 cells/well) in DMEM supplemented with 10% FBS. Hemsleya amabilis extract was added to the cultures at concentrations of 0, 25, 50, 100, 150, 200, and 300 Ag/ml, followed by an incubation. The cultures were maintained at 37 jC for 72 h. The cells were harvested by trypsinization, stained with trypan blue and the viable cells were counted. Hemsleya amabilis extract induced tumor cell death (n = 3).
To understand how Hemsleya amabilis extract induced cell death, we examined cell morphology affected by this compound. U87 astrocytoma cells were treated with or without Hemsleya amabilis extract. We demonstrated that cell spreading and elongation of U87 astrocytoma cells was severely affected by Hemsleya amabilis treatment at a concentration of 25 Ag/ml for 24 hours (Fig. 4B) compared with the untreated control (Fig. 4A). Treatment with 25 mg/ml of Hemsleya amabilis extract for 48 hours reduced the number of cells attached to plates (Fig. 4C). These results suggest that Hemsleya amabilis
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Fig. 4. Hemsleya amabilis extract inhibited tumor cell spreading. U87 cells were seeded on 6-well tissue culture plates (2 105 cells/well) DMEM supplemented with 10% FBS in the absence and presence of Hemsleya amabilis extract and incubated for 24 h at 37 jC. Without Hemsleya amabilis treatment, U87 cells spread well on the plates with extended processes (A). In the presence of Hemsleya amabilis extract (25 Ag/ml) for 24 hours (B) and 48 hours (C), cell spreading was greatly inhibited: most of the cells rounded up.
extract inhibited the interactions of cells with extracellular matrix. On the other hand, Jurkat cells, which could grow anchorage-independently, died at significantly higher concentrations compared with U87 and MDA-MB-231 cells (data not shown).
Fig. 5. Hemsleya amabilis extract promotes apoptosis of astrocytoma cells. U87 cells were seeded on 6-well tissue culture plates (2 105 cells/well) in DMEM supplemented with 10% FBS in the presence of Hemsleya amabilis extract at the final concentrations of 0, 25, and 50 Ag/ml. The cultures were maintained at 37 jC for 72 h. The cells were harvested by trypsinization, stained with annexin V-FITC, and analyzed on flow cytometry. The percentages indicate the proportion of apoptotic cells in the whole population.
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To investigate if the cells died necrotically and/or apoptoticly, we examined cell apoptosis after Hemsleya amabilis extract treatment. U87 cells were treated with or without Hemsleya amabilis extract. Apoptotic cells were detected using an annexin V-FITC apoptosis marker and quantified by flow cytometry. Massive cell apoptosis was found in Hemsleya amabilis extract treated cells (Fig. 5). Comparing with cell viabilities (Fig. 4), higher proportions of cells had undergone apoptosis. This was due to the high sensitivity of annexin V in apoptosis detection. Both the cell death and apoptosis were in a dosage-dependent manner (Fig. 3 and Fig. 5).
Discussion Hemsleya amabilis is an herb widely growing in many provinces in China. Hemsleya amabilis has been used in the treatment of many infectious illnesses. In recent years it has been used clinically to treat cancers and has been demonstrated an ability to greatly inhibit tumor development and growth with limited side effects [25]. However, the mechanism underlying its anti-tumor effect is not clear. In this study, we investigated the effect of Hemsleya amabilis extract on cell activities. We demonstrated that Hemsleya amabilis extract had significant effects on inhibiting tumor cell growth, colony formation and inducing tumor cell apoptosis. Inhibiting tumor growth has been a continuous effort in cancer treatment. A reduction in cell growth and an induction in cell death are two major means to inhibit tumor growth. In this study, we demonstrated that, at low concentrations, Hemsleya amabilis extract caused significant inhibition of growth in U87 astrocytoma cell line, MDA-MB-231 breast cancer cell line and Jurkat cells. These cells were derived from different tissue sources. The inhibitory effect of Hemsleya amabilis extract on cell growth implies that this compound may have a general function in anti-tumor cell growth. This is not unexpected, since cancer cells have developed the capacity of increased proliferation through a variety of growth signal pathways. This includes elevated external growth factors, increased intracellular matrix signal via integrin [30], and Ras protein mutation-derived constitutive mitogenic signals [31], resulting in growing neoplasm, that causes destruction and atrophy of the surrounding tissue and adjacent organs. In a specific tumor, one pathway may play a more important role than the others. Hemsleya amabilis extract may act on more than one pathway. Nevertheless, we did observe different sensitivities of tumor cells to the growth inhibitory effect of treatments of Hemsleya amabilis extract. Jurkat cells, for example, which were poorly anchorage-dependent in growth and survival, were less sensitive to the treatments as compared with U87 astracytoma cell line and MDA-MB-231 breast cancer cells, which were anchoragedependent in growth. This is coincident with the findings that Hemsleya amabilis extract inhibited U87 cell spreading. These results suggest that Hemsleya amabilis extract may interfere with the interaction of tumor cells with ECM, or signals of the interaction. To develop a tumor, cancer cells have to form a nude in vivo as the first step. We investigated the effect of Hemsleya amabilis extract on colony formation in vitro. Cancer cells are able to form colonies in soft agarose gel, and this was confirmed in the controls, in which no Hemsleya amabilis extract was added to the soft agarose gel cultures. The addition of Hemsleya amabilis extract inhibited colony formation completely. This result may explain the anti-cancer effect of Hemsleya amabilis extract in clinical application. Normal organ development is controlled by a balance between cell proliferation and apoptosis [19]. In cancer, the balance between proliferation and programmed cell death is disturbed. There is strong
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evidence that tumour growth is a result of uncontrolled proliferation and reduced apoptosis [32,33]. Cancer cells have an acquired capability to evade apoptosis through a variety of ways [32,33]. Inducing tumor cell apoptosis is an ideal way to kill cancer cells. In this study, when different human tumor cells were treated with Hemsleya amabilis extract, they underwent apoptotic death. However, different sensitivities were observed among different tumor cells. In a similar fashion, adherent astrocytoma and breast cancer cells are more sensitive to Hemsleya amabilis extract, compared with anchorageindependent Jurkat cells. Apoptosis is modulated by anti-apoptotic and pro-apoptotic effectors, which involve a large number of proteins [19,32,33]. Hemsleya amabilis extract-induced tumor cell apoptosis may involve an array of mechanisms. However, the difference in sensitivity to Hemsleya amabilisinduced apoptosis between the adherent and non-anchorage-dependent cells suggest that Hemsleya amabilis extract may inhibit ECM-derived cell survival signals, which play an important anti-apoptotic role [34,35]. Furthermore, the fact that the extracts induce non-anchorage dependent Jurkat cell apoptosis at increased concentrations suggests that other mechanisms are involved. Our results suggest that the anticancer effect of Hemsleya amabilis is a combination of its effects in inhibiting tumor cell growth and inducing tumor cell apoptosis. It thus has a great advantage over other types of treatments such as chemotherapy and more recently, hormonal treatments. Most of the chemotherapy treatments fail due to drug resistance. As an herb extract, Hemsleya amabilis contains a variety of compounds that may act on different pathways of tumor cell growth and survival. The molecular mechanisms underlying these effects await further investigation. Acknowledgements This work is supported by the University of Hong Kong. References [1] Pezzuto JM. Plant-derived anticancer agents. Biochemical Pharmacology 1997;53(2):121 – 33. [2] DiPaola RS, Zhang H, Lambert GH, Meeker R, Licitra E, Rafi MM, Zhu BT, Spaulding H, Goodin S, Toledano MB, Hait WN, Gallo MA. Clinical and biologic activity of an estrogenic herbal combination (PC-SPES) in prostate cancer. New England Journal of Medicine 1998;339(12):785 – 91. [3] Tiwari RK, Geliebter J, Garikapaty VP, Yedavelli SP, Chen S, Mittelman A. Anti-tumor effects of PC-SPES, an herbal formulation in prostate cancer. International Journal of Oncology 1999;14(4):713 – 9. [4] Darzynkiewicz Z, Traganos F, Wu JM, Chen S. Chinese herbal mixture PC SPES in treatment of prostate cancer (review). International Journal of Oncology 2000;17(4):729 – 36. [5] Duong Van Huyen JP, Sooryanarayana, Delignat S, Bloch MF, Kazatchkine MD, Kaveri SV. Variable sensitivity of lymphoblastoid cells to apoptosis induced by Viscum album Qu FrF, a therapeutic preparation of mistletoe lectin. Chemotherapy 2001;47(5):366 – 76. [6] Sakamoto S, Kudo H, Kuwa K, Suzuki S, Kato T, Kawasaki T, Nakayama T, Kasahara N, Okamoto R. Anticancer effects of a Chinese herbal medicine, juzen-taiho-to, in combination with or without 5-fluorouracil derivative on DNA-synthesizing enzymes in 1,2-dimethylhydrazine induced colonic cancer in rats. American Journal of Chinese Medicine 1991;19 (3 – 4):233 – 41. [7] Prozesky EA, Meyer JJ, Louw AI. In vitro antiplasmodial activity and cytotoxicity of ethnobotanically selected South African plants. Journal of Ethnopharmacology 2001;76(3):239 – 45. [8] Kawada M, Ohno Y, Ri Y, Ikoma T, Yuugetu H, Asai T, Watanabe M, Yasuda N, Akao S, Takemura G, Minatoguchi S, Gotoh K, Fujiwara H, Fukuda K. Anti-tumor effect of gallic acid on LL-2 lung cancer cells transplanted in mice. Anticancer Drugs 2001;12(10):847 – 52.
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