Cytotoxic activity of Hypericum perforatum L. on K562 ...

66 PHYT OTHERAPY RESEARCH Phytother. Res. 18, 66–72 (2004) Published online in Wiley InterScience (www.interscience.wiley.com). G. ROSCET TI ET ALDOI.: 10.1002/ptr.1369

Cytotoxic Activity of Hypericum perforatum L. on K562 Erythroleukemic Cells: Differential Effects between Methanolic Extract and Hypericin Gianna Roscetti1*, Ornella Franzese2, Alessandro Comandini2 and Enzo Bonmassar2 1

Chair of Human Physiology, Department of Neuroscience, School of Medicine, University of Rome Rome, Italy Chair of Pharmacology, Department of Neuroscience, School of Medicine, University of Rome, Rome, Italy

2

The influence of a methanolic extract of Hypericum perforatum L. and of purified hypericin has been comparatively tested on the growth of a human erythroleukemic cell line (K562). After 1 h exposure to increasing concentrations (as hypericin content) of both agents in the dark, leukemic cells were grown for 24 h and 48 h. The effects on cell growth were determined by viable cell count, flow cytometry analysis and fluorescence microscopy. Our data show that purified hypericin has only a weak inhibitory effect on cell growth and no effect in inducing apoptotic cell death. In contrast, the Hypericum flower extract shows a significant concentration-dependent and long-lasting inhibition of cell growth, and induces apoptotic cell death. This work confirms the interesting role of Hypericum perforatum L. in cancer therapy and strongly supports the hypothesis that agents, other than hypericin, present in the total extract can impair tumor cell growth acting separately or in a combined manner. Copyright © 2004 John Wiley & S ons, Ltd. Keywords: Hypericum extract; apoptosis; cell growth; cancer therapy; hypericin. Copyright © 2004 John Wiley & Sons, Ltd.

INTRODUCTION

Total extract of Hypericum perforatum L. (Hypericaceae) is largely used in the treatment of moderate and mild depression and it is under study for antiviral and antitumor activities. Actually, Hypericum contains several compounds which may have interesting pharmacological properties such as hypericins (a group of condensed anthraquinones found almost exclusively in this genus), a number of flavonoids, condensed tannins and hyperforin. Recently, hypericin has been found to be highly active against tumor growth both in vivo and in vitro, only after photoactivation with either visible or UV light (Thomas and Pardini, 1992; Vandenbogaerde and de Witte, 1996). Hypericin is thus considered a very interesting compound for tumor photodynamic therapy (Fox et al., 1998; Lavie et al., 1999). Furthermore, its anti-tumor activity has been shown to be partially related to the inhibition of protein kinase C (PKC) (Takahashi et al., 1989; Samel and de Witte, 1996) and free radical induction (Thomas and Pardini, 1992; Miccoli et al., 1998). Finally, the ability of photoactivated hypericin to induce necrosis and apoptosis has been related to its action on the release of mitochondrial cytochrome c and the activation of procaspase-3, two

* Correspondence to: G. Roscetti, Chair of Human Physiology, Department of Neuroscience, School of Medicine, University of Rome-Tor Vergata, via Montpellier n. 1 00133 Rome, Italy. Tel.: (+39)0672596856. Fax: (+39)0672596855. E-mail:[email protected] Contract/grant sponsor: Ministry of Education, University and Research of the Italian Government.

Copyright © 2004 John Wiley & Sons, Ltd.

characteristic events of the cell death program (Vantieghem et al., 1998). The flavonoids found in Hypericum are common in the plant kingdom, and some of them have been recognized to possess anti-tumor activity. In particular, quercetin and apigenin have been found to inhibit cell growth and induce apoptosis in a number of tumor cell lines (Azuma et al., 1995; Kuo, 1996; Makita et al., 1996; Richter et al., 1999) and to increase caspase activity and cytochrome c release (Wang et al., 1999). However, these flavonoids do not influence the growth and apoptosis of a murine B16 melanoma 4A5 cell line (Iwashita et al., 2000). Many data show that the major constituent responsible for the antidepressant activity of Hypericum extract is hyperforin, an acylphloroglucinol compound (Chatterjee et al., 1998; Wonnemann et al., 2000; Barnes et al., 2001). No data are presently available concerning possible effects of hyperforin against tumor cell growth. However, intriguingly, in a study on the antiinflammatory properties of Hypericum extracts, hyperforin was found to inhibit the proliferation of peripheral blood mononuclear cells (PBMC) (Schempp et al., 2000). Condensed tannins present in Hypericum are a heterogeneous class of oligomeric polyphenols (proanthocyanidins) common in plants and known to be natural antioxidants. They have been shown to reduce free radical formation, oxidative stress, lipid peroxidation, and capillary fragility and also to act against bacterial and viral infections, inflammation s , allergies and tumors (Ye et al., 1999; Fine, 2000). The available data are often contradictory, however, probably due to the great heterogeneity of this class of compounds. Interestingly, tannins have been found to inhibit growth and induce apoptosis in tumor cells, whereas non-transformed cells are sometimes stimulated to grow (Ye et al., 1999; Phytother. Res

CYT OTOXIC ACTIVIT Y OF HYPERICUM PERFORATUM L. Received 16 January 2003. 18, 66–72 (2004) Accepted 24 April 2003

Sakagami et al., 2000; Yoshida et al., 2000; GomezCordoves et al., 2001). The aim of the present work is to compare the cytotoxic properties of the total extract of Hypericum with those of nonphotoactivated hypericin in order to detect the activity of other compounds capable of interfering with cell growth. Actually, it is reasonable to hypothesize that a number of different compounds present in the Hypericum extract may interact with distinct cell growth mechanisms, thus producing antiblastic effects more evident than those detectable with each single agent.

MATERIALS AND METHODS Drug preparation. Plants were collected locally, while in flower and identified as Hypericum perforatum L. (Baroni, 1969). A voucher specimen has been deposited in our laboratory at the Department of Neuroscience, School of Medicine, University of Tor Vergata, Rome, Italy (GR6.99RcPr). After being dried in a cool, shaded, airy place, the flowers were minced and stored, protected from light and humidity. Subsequently, 0.5 g of herbal drug (minced dried flowers) was extracted with methanol (HPLC grade, Merck D-6100 Darmstadt, F. R. Germany) by continous rotation for 60 min. The extraction was performed three times, that is, as long as the red color due to hypericins continued to be released. After centrifugation at 1160 × g for 10 min (Centrifuge Beckman J2-21), supernatants were pooled and evaporated until dry. All drying procedures were made rapidly (little volumes at a time of volatile organic solvents), in a rotating system under reduced pressure (Speed Vac SVC 100-Savant, Instruments Inc., Farmingdale, NY, USA) and without heating. This dried extract (HyEx) and purified hypericin (Hyp) (AlexisItalia, Vinci-Florence, Italy) were both dissolved in ethanol (HPLC grade, Merck) and used for the spectroscopic analyses. The various hypericins present in the extract, which are spectroscopically indistinguishable from each other, were estimated as hypericin by the absorbance peak at 590 nm (molar extinction coefficient of 46 000 M −1 cm−1 in ethanol) (Liebes et al., 1991; Wagner and Bladt, 1994). HyEx and Hyp were diluted to a concentration of 1 µg/ml of hypericin each and UV/ Vis spectra of both solutions were recorded between 250 nm and 700 nm with a Cary 219 recording spectrophotometer (Varian, PaloAlto, CA USA). The solutions were then dried, and stored at −20 °C until use. Cell culture. K562 human erythroleukemic cells, obtained from ‘American Type Culture Collection’ (ATCC, Maryland, USA, code CCL 243), were grown in RPMI 1640 (Hyclone, Europe Ltd, UK) supplemented with 10% heat-inactivated fetal calf serum (Hyclone Europe Ltd., Cramlington, UK) 2 mM Lglutamine and 100 UI/ml of penicillin/streptomycin (Flow Laboratories, McLean, VA, USA). The cells were maintained in a humidified incubator with 5% CO2 at 37 °C. The growth medium was replaced periodically, and again just before the test. Experimental procedure. 5 × 105 cells/ml were suspended in RPMI and exposed to HyEx and to Hyp at concentrations of 0.5, 1.0, 2.5, and 5.0 µg/ml of hypericin content or to vehicle alone (control cells). Both treated and untreated cells Copyright © 2004 John Wiley & Sons, Ltd.

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were maintained at 37 °C and 5% CO2, in the dark, for 1 h, then rinsed twice and suspended in the growth medium. Finally, 5 × 105 cells/ml were transferred in triplicates into 24-well plates, and incubated for 24 or 48 h. Cell growth and viability assessment. After incubation, cell growth and viability were assessed by phase contrast microscopy after the addition of 1% trypan blue. The significance of the results was assessed using Student’s ttest. Fluorescence microscope assay. 100 µl per well (i.e. 10% of the cells) were mixed with formaldehyde (4% final concentration) for 10 min. After 10-min centrifugation at 400 × g, the pellets were dried on glass microscope slides, and examined with an inverted fluorescence microscope (Leitz, Laborlux 12) using a 50 × water immersio n objective (total magnification 500 ×). All the triplicates were carefully observed. To identify the red fluorescence due to hypericin, cells were observed at an excitin g wavelength of 475 nm. The state of the DNA – spread, compacted, or fragmented – was determined by staining the cells with bisbenzimide, Hoechst 33342 (HO, Sigma Chemical Co. St Louis, MO, USA). HO was dissolved in water (1 µg/ml final concentration) and used according to published methods (Maciorowski et al., 1998; Choi and Jung, 1999). Briefly, the cells dried on glass slides were exposed to the DNA dye for 10 min in the dark at room temperature and were then washed gently. After microscopic observation at an exciting wavelength of 340– 380 nm, cells were photographed with an upside affixed camera (Wild MPS 51, Switzerland). HO, which is generally recognized as a marker of early apoptosis, enters the cells both through intact and damaged membranes and stains DNA from blue to bright white, depending on the degree of chromatin condensation. Flow cytometric analysis. DNA fragmentation was analyzed by flow cytometric assay. After 24 and 48 h the triplicates were pooled and the cells washed with PBS, fixed with 1% formaldehyde, and incubated, first 1 h at −20 °C with 70% ethanol and then, after washing, with a propidium iodide (PI, Boehringer Mannheim, Mannheim, Germany) solution (sodium citrate 0.1%, RNAse 20 µg/ml, PI 50 µg/ml, NP40 0.3%). To visualize DNA fragmentation after propidium iodide staining, 10 000 cells per sample were analyzed by a FACScan flow cytometer (Becton Dickinson, Erenbodegem-Aalst, Belgium). The relative DNA content, based on red fluorescence levels, was calculated with ‘Lysis’ program (Becton Dickinson).

RESULTS Spectroscopic profiles The hypericins present in the extract include main ly pigments such as hypericin, pseudohypericin and protopigments with the same UV/Vis spectra (Kurth and Spreemann, 1998). Since hypericin is generally recognized as the most active of these pigments, we used purified hypericin (Hyp) as reference compound. However, the extract (HyEx) also contains a second group of compounds showing main absorbance peaks in the 250–450 nm range, including hyperforin, condensed tannins and a number of Phytother. Res. 18, 66–72 (2004)

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flavonoids (in particular quercetin, apigenin and derivatives). Fig. 1 shows the UV/Vis spectrum of Hy Ex (A) compared with Hyp (B) at 1 µg/ml of hypericin each. Total extract causes a greater cell growth inhibition To investigate the influence of Hypericum on cell growth we exposed K562 cells to the agents under investigation. K562 cell line is a human chronic myelogenous leukemia suitable not only as a target susceptible to natural killer effector cells, but also for screening anticancer agents as a target of a number of antitumor compounds, including plant extracts (Yan et al., 2002). It is sensitive to therapeutics, multi drug resistance (MDR) proteins are not overexpressed but it is easy to induce their expression, when needed (Loetchutinat et al., 2003). Further, it is possible to induce it to differentiate into erithrocyte, granulocyte or monocyte series allowing further pharmacological studies. Preliminary experiments with rat glial tumor cell line (C6) and human lymphocytic leukemic cells containing HTLV1 (MT2), and with different extracts (ethanolic, ether/ethanolic) gave results consistently similar to those reported here (data not shown). Both Hyp and HyEx were suspended in RPMI at final concentrations of 0.5, 1.0, 2.5 and 5.0 µg/ml of hypericin, as described in Materials and Methods. These concentrations were chosen on the basis of preliminary tests showing that extract containing more than 5.0 µg/ml hypericin – and proportionally larger amounts of the other compounds – was extremely toxic for the cells. These concentrations are also within the range of the pharmacologic activity of Hyp in other experiment al models (Miccoli et al., 1998; Vantieghem et al., 1998; Lavie et al., 1999). Viable cell counts are summarized

h, allowing a partial recovery of cell growth. It is noteworthy that, at 0.5 µg/ml concentration, both cells exposed to HyEx and those exposed to Hyp showed a similar growth rate either after 24 h and 48 h. At the concentration of 1.0 µg/ml, HyEx inhibited cell growth significantly more than Hyp, after 48 h. This different effect increased proportionally at 2.5 and 5.0 µg/ml concentrations both at 24 h and 48 h. These results suggest that the different effects of the two types of treatment must be due to one or more compounds, present in HyEx, distinct from hypericin and able to produce additional growth-inhibiting effects in a concentration-dependent manner.

Effect of the total extract on programmed cell death The next step was to test whether the additional inhibitory effect on cell growth of the unknown compound(s) was due to the activation of necrotic and/or apoptotic mechanisms. By means of FACScan analysis we observed the presence of apoptotic cells, which was confirmed by direct fluorescence microscope observation (see below). Fig. 3 shows the results of FACScan analysis. The peak value occurred with HyEx at a concentration of 2.5 µg/ml of hypericin (at 24 h, 36.3% more than the control). Hyp did not show apoptotic effect both after 24 h (apoptosis was only 5% greater than the control at the highest concentration) and

Figure 2. K562 cell growth at 24 h and 48 h culture after treatment. Before culture, cells were exposed to the concentrations indicated (expressed as hypericin content) or to medium alone, for one hour in the dark. Bars represent counts of viable cells (percent of control) exposed to HyEx (black bar) and Hyp (striped bar). A representative experiment run in triplicate is reported. Means ± SD are shown. * p < 0.01 vs control. Figure 1. UV/Vis spectrum of Hypericum extract (HyEx) containing 1 µg/ml of hypericin ((A) and black line in (B)) compared to the spectrum of 1 µg/ml of pure hypericin (Hyp) (grey line in (B)). Determinations were performed in ethanol (hypericin, ε 590 = 46 000).

in Fig. 2 where the different effects of HyEx and Hyp can be seen. After 24 h cells exposed to HyEx showed a strong cell growth inhibition in a concentrationdependent manner. At 2.5–5.0 µg/ml, growth inhibition was similar or even greater at 48 h with respect to that detectable at 24 h. This indicates that HyEx possesses a strong and long-lasting suppressive effect on K562 cells. On the other hand, Hyp was much less active than HyEx on leukemic blasts, showing significant suppressive activity at all concentrations at 24 h, but not at 48 Copyright © 2004 John Wiley & Sons, Ltd.

after 48 h (see values given in brackets). The effects of the two agents under study on cell growth were then directly observed by means of HO staining. Fig. 4 shows a series of pictures taken at the inverted fluorescence microscope of treated and untreated cells after 48 h culture. At this exciting wavelength (340–380 nm) the fluorescence due to hypericin content is completely attenuated, while the HO dye is clearly visible as white-blue fluorescence (white-grey in photographs). HO staining appears as bright white when chromatin is compacted. Microscopic observation of the triplicates showed at least five sub-populations and confirmed the results of FACScan analysis. In the first photo, untreated cells appear in normal condition with clearly visible Phytother. Res. 18, 66–72 (2004)

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nuclei and faintly stained chromatin and cells in mitosis are frequent (subpopulation 1). Cells exposed to the lowest concentration of HyEx (i.e. 0.5 µg/ml of hypericin content) are very similar to the controls, although some nuclei appear more intensely stained (subpopulation 2). The situation changes when cells are exposed to the next higher concentration (1 µg/ml). In this case, a sharp increase of shrunken cells with bright white apoptotic bodies due to chromatin condensation (subpopulation 3) along with rare normal cells can be seen. At the next concentration of HyEx (2.5 µg/ml), apoptotic cells with picnotic and fragmented nuclei are clearly visible (subpopulation 4). The photograph at 5.0 µg/ml concentration shows apoptotic and necrotic cells; cells in necrosis can be recognized by widespread chromatin weakly stained by HO and cell swelling with total loss of nuclear membrane integrity (subpopulation 5) (Choi and

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Jung, 1999). In contrast, the photograph relative to the 5.0 µg/ml concentration of Hyp shows lack of apoptotic and necrotic effects of this drug as the control cells (subpopulation 1). In the same way, after 24 h culture, cells exposed to Hyp were similar to control cells at each concentration tested while HyEx treated cells showed concentration-dependent apoptotic and necrotic effects (data not shown).

DISCUSSION The aim of this work was to examine the cytotoxic activity of compounds present in Hypericum perforatum L. that might have a role in cancer therapy. We studied the effect on the growth of K562 leukemic cells of a

Figure 3. Representative FACScan analysis of apoptotic K562 cells. The diagrams refer to cells exposed (1 h in the dark) to the indicated concentrations of HyEx (upper row), and Hyp (middle row), as well as non-treated cells (bottom), after 24 h culture. Fragmented DNA is shown under the M1 gate on the left of the G0/G1 cell cycle peak. Percent numbers represent the events under t he gate, and the respective values after 48 h culture are also indicated (in brackets). Ten thousand cells from each sample were analysed, as described in Materials and Methods.


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Figure 4. Apoptosis and necrosis of K562 cells exposed to HyEx and to Hyp (1 h in the dark) after 48 h culture. Cells, treated and untreated, were dried on glass slides, stained with Hoechst 33342 and observed at an exciting wavelenght of 340–380 nm. Upper left: untreated cells (CTRL). Upper right: cells exposed to Hyp. Centre left to lower right, cells exposed to increasing concentration of HyEx. Concentrations of hypericin content are indicated. Representative photographs, chosen from each observed triplicate, are shown.

methanolic extract (HyEx) of Hypericum perforatum L. flowers, comparing it with purified hypericin (Hyp). Hypericum contains a number of active agents which may interfere with cellular dynamics (e.g. Fox et al., 1998; Ye et al., 1999; Wang et al., 1999; Schempp et al., 2000). The sample of Hypericum we analysed contained 0.12% total hypericins with respect to the overall dried weight, found almost exclusively in the flower heads. In 1 h reaction time hypericins enter into the cells in large amounts, and remain in the cytoplasm for a long time. Even after 48 h culture, cells show a cytoplasmic (not nuclear) red fluorescence proportional to hypericin concentration (data not shown). Thus it is possible that hypericin as well as other compounds were confined to the mitochondrial and reticular compartments of the cell, from where they could activate metabolic patterns leading to growth inhibition and/or to programmed cell death (such as PKC inhibition, formation of oxygen radicals, caspase activation or


cytochrome c release) (Vantieghem et al., 1998; Wang et al., 1999). On the other hand, it is possible to hypothesize that non-fluorescent compounds (e.g. condensed tannins) could reach the nucleus and induce DNA fragmentation directly or indirectly. As for the action of HyEx, we observed a strong inhibitory effect on cell growth. This activity is concentration dependent and is observable from hypericin concentrations of 0.5 µg/ml (i.e. 1.0 µM). By contrast, Hyp has a weaker, though still significant, inhibitory effect on cell growth. In any case, the suppressive effects of Hyp are detectable mainly after 24 h, followed by marked recovery of tumor cell growth at 48 h. Further, when HyEx was used at concentrations of 1.0 to 5.0 µg/ml, essentially no cell growth recovery could have been observed at 48 h. The suppressive effect of Hyp could be due mainly to mechanisms such as PKC inhibition (Takahashi et al., 1989; Samel and de Witte, 1996) and/or

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formation of oxygen radicals (Thomas and Pardini, 1992; Miccoli et al., 1998) producing a short-term effect that disappears after 48 h culture. Actually, it is conceivable that both biochemical lesions could be reversed by repair mechanisms associated with cell metabolism. For examp le, drug-mediated induction of reactive oxygen species is generally followed by increased expression of antioxidant enzymes such as superoxide dismutase, which, in turn, can contrast cell damage produced by oxygen radicals (Kong and Lillehei, 1998). In the case of HyEx, additional compounds must play a role, because at 1.0– 5.0 µg/ml of hypericin content, other more efficient, concentration-dependent and long-lasting mechanisms appear to be activated. Fragmented DNA, apoptotic bodies and necrosis are important events at these concentrations, as seen at FACScan analysis and microscopic observation. In conclusion, non-photoactivated hypericin has a weak, short-term cell growth inhibition effect with no apparent Azuma Y, Onishi Y, Sato Y, Kizaki H. 1995. Effects of protein tyrosine kinase inhibitors with different modes of action on topoisomerase activity and

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concentration-effect relationship, and no effect on cell death programs, at least at concentrations up to 5.0 µg/ml. On the other hand, the methanolic extract, at corresponding concentrations of hypericin content, produces significant, long-lasting and concentration-dependent cell growth inhibition due to the activation of apoptotic and necrotic mechanisms. However, whether these effects are to be attributed to a single compound or to several compounds, or to the combined activity of a number of compounds, will be the subject of further investigation.

Acknowledgements T his work was supported in part by a grant obtained by Gianna Roscetti and in part by a grant obtained by Enzo Bonmassar, both from the Ministry of Education, University and Research of the Italian Government. Richter M, Ebermann R, Marian B. 1999. Quercetin-induced apoptosis in colorectal tumor cells: possible role of EGF receptor signaling.

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