The Lipophilic Extract of Hypericum perforatum Exerts Significant Cytotoxic Activity Against T24 and NBT-II Urinary Bladder Tumor Cells

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Plant material
• Solutions, extracts and fractions preparations
• Quantification of herb’s main biological constituents
• Cell proliferation assay
• TUNEL assay
• Results
• Discussion
• Acknowledgements
• References

Abstract

Hypericum perforatum L. (St. John’s wort) is a medicinal plant used for many pathologies, especially for the treatment of mild to moderate depression. In the present study we have investigated the cytotoxic activity of the locally collected (Epirus region) Hypericum perforatum L. against cultured T24 and NBT-II bladder cancer cell lines. The lipophilic extract of the herb, prepared using petroleum ether, induced apoptosis displaying LC₅₀ values at concentrations as low as 4 and 5 μg/mL. A fraction of this extract displayed 60 % cell growth inhibition at a concentration of 0.95 μg/mL. Evaluating the importance of various biologically active components of the extract, it was found that hypericins (hypericin, pseudohypericin, etc.) were identified only in the methanolic (lipophobic) extract of the herb, and not in the active lipophilic extract. In addition, hyperforin concentrations in the lipophilic extract and its most active fraction, were 0.94 μg/mL, and 0.17 μg/mL, respectively, while the active cytotoxic concentration of pure hyperforin appeared in the range of 1.8 μg/mL - 5.0 μg/mL. Therefore, pure hyperforin does not seem to contribute significantly to the cytotoxicity activity. Chlorophylls were identified in low, not significantly different, concentrations in all extracts and fractions and were not correlated to the biological activity.

Owing to the combination of significant cytotoxic activity, natural abundance and low toxicity, the lipophilic extract of Hypericum perforatum holds the promise of being an interesting, new, antiproliferative agent against bladder cancer that deserves further investigation.

Abbreviations

HP:Hypericum perforatum L.

AS:aqueous solution

MS:methanolic solution

ME:methanolic extract

NPMF:non polar methanolic fraction

PMF:polar methanolic fraction

PEE:petroleum ether (lipophilic) extract

PEE (1,2,3,4,5,6,7):petroleum ether extract's fractions

PBS:phosphate buffered saline

DMSO:dimethyl sulfoxide

Introduction

Bladder cancer, related to many environmental carcinogens and tobacco smoke, is a major disease with over 50,000 new cases and approximately 10,000 deaths per year in the U.S.A. alone [1]. The variable morphology, natural history and prognosis demonstrate that transitional cell carcinoma (TCC) of the bladder is not a single disease, but occurs generally in two major categories, namely superficial and invasive. More than 50 % of new bladder cancer cases are superficial. These types of tumors are considered to have low invasive potential with a progression rate to invasive cancer of 15 to 20 %. These types of cancer will recur within 12 months after resection in 50 to 90 % of the patients who have been treated [2].

The necessity for early adjuvant treatment and mainly intravesical instillations of immuno- or chemotherapeutics in the management of high-risk superficial bladder tumors is recognized globally and is based on the hope that this treatment, by altering the neoplastic potential of the urothelium [3], [4], [5], may reduce the risk for recurrence and progression, which are the most worrisome problems in managing this type of cancer [6]. Different modalities, such as photodynamic therapy, have also been tried for the same disease, and have shown encouraging results [7].

Hypericum perforatum L. (HP) or St. John’s wort, has been used for centuries for the treatment of burns, bruises, swelling, inflammation and anxiety, as well as bacterial and viral infections [8]. The use of its top flowering parts was originally documented by the ancient Greek medical herbalists Hippocrates, Theophrastus, and Dioscorides [9]. Locally, it is traditionally used both externally (as a cream), and internally (as a tea) with many therapeutic applications [10]. In the last two decades, anti-inflammatory, antimicrobial, antiviral and antidepressant activities of the herb have been attributed to the total extract or single constituents [11], [12], [13], [14]. Today, HP is becoming increasingly popular for the treatment of mild to moderate depression, due to its low toxicity, and effectiveness [15].

A qualitative analytical composition of the herbal drug includes among others naphthodianthrones, flavonoids, biflavones, phloroglucinols, volatile oils, xanthones, phenolic acids, chlorophylls, sterols, vitamins, xanthophylls, coumarins etc. [16]. The contents of the extracts vary significantly in plants collected from different locations, and depend on the stage of development of the plant [17]. The antidepressant activities of the herb were first attributed to hypericin and pseudohypericin and to flavonoids, but recent pharmacological and clinical results focus on hyperforin as the main active ingredient of the extract [18], [19], [20]. The structures of these pharmacologically active chemicals are shown in Fig. [1] [21].

Anecdotal clinical observations, at the local level of Epirus Greece, regarding the probable preventive role of the aqueous HP extract against superficial bladder cancer have been recorded during the last five years. Based on these observations, we initiated this study, testing first the effectiveness of the aqueous herbal solution since the locals consume it systematically. The study has been designed in order a) to find the most active extract of HP from Epirus against selected bladder cancer cell lines, b) to explore the possible effect of the herb’s extract(s) against the bladder cancer cell lines.

Materials and Methods

Plant material

The fresh aerial parts of the HP from Epirus were collected in Ioannina (Epirus region), Greece, in June 2003. The voucher specimen has been deposited at the Agricultural University of Athens, Greece (N. 1022). The plant material was dried, pulverized and stored until used (2 kg).

Solutions, extracts and fractions preparations

Fig. [1] of the Supporting Information shows the extraction procedures used for the processing of the dried plant. Herbal solutions were first prepared as ”boiled tea”, aqueous solution (AS) using phosphate-buffered saline (PBS), and methanolic solution. The methanolic extract (ME) of the herb was prepared in 11 % yield, and it was fractionated using liquid/liquid extraction with petroleum ether and methanol [polar methanolic fraction (PMF) in 90 % w/w, and the non polar-lipophilic methanolic fraction (NPMF) in 10 % w/w]. The petroleum ether (lipophilic) extract of the herb (PEE) was prepared as well, in 2 % w/w only, and was subjected to fractionation by column chromatography using 10 % methanol in isocratic dichloromethane as eluent. The seven fractions collected, with their percentage contribution to the total amount are shown in Fig. [1] of the Supporting Information.

Quantification of herb’s main biological constituents

The quantitative determination of the main biological constituents in each extract and fraction of the plant was measured in mg/100 mg extract, or fraction, and is shown in Table [1]. The determination of the total naphthodianthrones content was carried out by following the spectrophotometric procedure adapted by the U.S. Pharmacopoeia [22]. The total chlorophylls, chlorophyll a, and chlorophyll b contents in the plant extracts/fractions were determined according to the official AOAC spectrophotometric method [23]. For the HPLC analysis of hypericin, pseudohypericin and hyperforin in plant extracts, a Shimadzu RP-HPLC-UV-fluorescence system was used with a Hypersil ODS column (250 × 4.6 mm I.D., 5 μm, Thermohypersil-Keystone, Cheshire, UK) thermostatted at 30 °C. The mobile phase consisted of methanol-ethyl acetate-buffer phosphate pH 2.2 (95 : 25 : 5, v/v/v) and was run isocratically, at a flow rate of 0.3 mL/min.

Cell proliferation assay

The T24 human bladder cancer cell line was derived from a grade 3 human bladder tumor. The NBT-II rat bladder cancer cell line was derived from a male Wistar rat bladder tumor induced by N-butyl-N-(4-hydroxybutyl)nitrosamine. T24 cells were cultured in Dulbecco’s modified Eagle’s medium, 1 g/L D-glucose, 10 % fetal calt serum, supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM glutamine. NBT-II cells were cultured in minimum essential Eagle’s medium, 2 mM glutamine, 1 % non-essential amino acids, 1 mM NaP, 10 % fetal bovine serum, supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin and 2mM glutamine. The in vitro procedure followed in each experiment has been described earlier [24]. The results of all the experiments were expressed as the percentage reduction of number of cell colonies, when compared to the controls in each case and are shown in Figs. 2 - 4.

TUNEL assay

The early DNA breaks during apoptosis were assessed by the TUNEL assay using the ApopTag® Peroxidase In Situ Apoptosis Detection kit (Intergen, Serologicals company, USA). The apoptotic index was defined as the percentage of apoptotic cells in relation to the total number of cells.

Results

The inhibiting effects of the AS, MS, ME and fractions of HP from Epirus were first determined in the T24 human bladder tumor cell line. Fig. [2] A shows the cytotoxic activity of AS and MS. The MS was more effective than the AS (LC₅₀ = 1.1 μL/mL and 4.7 μL/mL, respectively). Fig. [2] B shows the effect on cell growth of the ME and its fractions. The ME induced cell growth inhibition with an LC₅₀ value of 18 μg/mL. Fractionation of the ME provided a less active fraction, the PMF with an LC₅₀ value of 29 μg/mL, and a more active fraction, the NPMF with an LC₅₀ value of 10 μg/mL. The effects of the ME and its fractions were also determined in the NBT-II rat bladder tumor cell line. The results are shown in Fig. [3] A. The ME induced cell killing with an LC₅₀ value of 13 μg/mL. The PMF was less active in this cell line also, with an LC₅₀ value > 40 μg/mL, and the NPMF fraction was more active, with an LC₅₀ value of 10 μg/mL. The effect of ME, PMF and NPMF in both cell lines follows the same trend.

The cytotoxic effect of the PEE was evaluated in the T24 and the NBT-II bladder tumor cell lines (Figs. [2] C and [3] B). In these two cell lines the extract exhibited an LC₅₀ value of 5 μg/mL in the T24 cells, and of 7 μg/mL in the NBT-11 cells. Significant 70 % cell killing was recorded at a concentration of 12 μg/mL in both cell lines.

In order to identify the fraction(s) of the PEE, responsible for the cytotoxic effect, the activities of its seven fractions (Fig. [1] in the Supporting Information) against the T24 cell line were evaluated. The results are shown in Fig. [4] A. The active concentration of PEE used was 6 μg/mL, since it resulted in a significant cell killing of 60 % (see Fig. [2] C). The concentrations used for each fraction, are proportionally related to the contribution of each fraction to the total amount collected through the column (in w/w). The concentrations of all fractions together add up to 6 μg/mL, which is equal to the concentration of the total extract PEE used in this experiment. Thus, the antitumor activity observed for each fraction is its actual contribution to the overall activity of the PEE. From all samples tested, only fraction PEE6 exhibited a significant inhibitory effect of 60 %, equal to the activity of the total extract PEE used. The active concentration of PEE6 was 0.95 μg/mL, compared with the corresponding 6μg/mL concentration of the total extract PEE.

NBT-II cells stained by the TUNEL method and visualized by light microscopy are shown in Fig. [2] of the Supporting Information. Dark brown staining indicates that apoptotic DNA breaks occurred in these cells. The tumor apoptotic index in the control cover slips was 0 %, while apoptosis rates were 90 % in the 12 μg/mL concentration of PEE-treated cells, 20 % in the 6 μg/mL concentration of PEE, and 10 % in the 3 μg/mL concentration of PEE. T24 cells were stained by the TUNEL method and were evaluated in the same manner. Apoptosis rates were 20 % in the 6 μg/mL concentration of PEE-treated cells, and 10 % in the 3μg/mL concentration of PEE. The apoptotic rates were well correlated (p = 0.000) to the cell killing rates recorded at the same concentrations, shown in Fig. [3] B, an indication that apoptosis is the main pathway by which the herb’s PEE extract induces cellular death.

The direct correlation between the difference in the activity of the methanolic/petroleum ether extracts (LC₅₀ of 29 μg/mL vs. 5 μg/mL), and their hyperforin concentrations (7.6 %/18.9 %), led to the investigation of the activity of hyperforin alone, in order to find out if this constituent is primarily responsible for the specific action. The antiproliferative potential of pure hyperforin was evaluated in the T24 cell line. As shown in Fig. [4] B, hyperforin induced an 80 % cell growth inhibition at a concentration of 2.5 μg/mL (LC₅₀ value at 1.9 μg/mL). The active concentration range of hyperforin was found to be between 1.8 μg/mL and 5.0 μg/mL. In our active PEE extract, and its active fraction PEE6, the concentration of hyperforin (LC₅₀ value) is 0.9 μg/mL, and 0.17 μg/mL, respectively (see Table [1], and Figs. [2] C and [4] A). Both concentrations are far below the hyperforin active concentration range of 1.8 μg/mL - 5 μg/mL.

Discussion

In the course of our in vitro experiments various solutions, extracts, and fractions of the HP herb were prepared and tested against two different bladder tumor cell lines (a human T24, and an animal NBT-II cell line). Since the methanolic solution appeared more active than the aqueous solution, we continued with the preparation of the methanolic extract, showing LC₅₀ values of 13 μg/mL and 18 μg/mL for the NBT-II and T24 cell lines, respectively. Hostanska et al. reported recently LC₅₀ values of the ethanolic extract of Hypericum perforatum between 1.9 mg/mL and 4.1 mg/mL, indicating probable differences between cell lines performance, plant characteristics, and extraction procedures used [25]. When we proceeded further with the fractionation of the methanolic extract we were able to show that the lipophilic portion was primarily contributing to this cytotoxic activity, compared to the lipophobic portion.

This observation prompted us to proceed with the preparation and testing of the petroleum ether extract. This extract proved to be significantly active, exerting a major growth inhibition on the cell lines used. The LC₅₀ values recorded were as low as 4 μg/mL, inducing cell killing by apoptosis. Fractionation of this extract, and testing in T24 cell line the seven fractions separated, indicated that the cytotoxicity of the total extract PEE is caused mainly by fraction PEE6 (Fig. [4] A). This fraction exhibited 60 % cytotoxicity, at the very low concentration of 0.95 μg/mL. The rest of the fractions showed very low to no activity.

In order to identify directly the constituent(s) responsible for the cytotoxic action, the main biologically active constituents of the herb were quantified in each preparation mentioned above. Total chlorophylls, chlorophyll a, and chlorophyll b were found in all extracts, and in fractions of the herb (Table [1]). Total hypericins, hypericin, and pseudohypericin were found in adequate amounts only in the methanolic extract (ME), and its polar fraction PMF. Hostanska et al., by studying the activity of pure hypericin and hyperforin towards human malignant cell lines, reported recently that their cytocidal effects on tumor growth inhibition in a synergistic manner make HP an interesting option in cancer treatment [26]. Furthermore, a recent report indicated that pure hyperforin is an active compound against a number of tumor cell lines [27]. Based on our observations concerning the activity of the lipophilic HP (PEE) extract, its fractions and its methanolic derivatives vs. the concentrations of constituents in these preparations, we draw the following conclusion.

The data concerning the methanolic extract’s (ME) activity, and naphthodianthrone concentration could probably lead to the conclusion that hypericins might play a role in the antitumor activity. However, the fact that the petroleum ether extract (PEE) showed a major antitumor activity without containing hypericins, indicates that their contribution seems to be non-significant. This is also apparent for the lipophilic fraction of the methanolic extract (NPMF), which is active without containing hypericins.

The data concerning the petroleum ether (PEE) extract/fraction activity and hyperforin concentration could sustain the indication that hyperforin is not one of the main active compounds of the herb, in our active extract. From the data in Table [1] and Fig. [4] A, it is apparent that even though the extract PEE and the active fraction PEE6 show a significantly different antitumor activity compared with the fractions PEE4 and PEE5, they all contain nearly the same amount of hyperforin (around 18 %). The concept that hyperforin is not one of the main active component(s) in our lipophilic extract, is also supported by the fact that the hyperforin concentrations in the active fractions of PEE, and in its most active fraction PEE6, are only 0.9 μg/mL and 0.17 μg/mL, respectively, which are below the active range of pure hyperforin in the same tumor cell line, shown in Fig. [4] B (1.8 μg/mL - 5.0 μg/mL).

The data concerning total chlorophylls, chlorophylls a and b, in all extracts and fractions indicate that they are present in small, or very small amounts in all preparations. The activity-concentration relationship of the chlorophylls in the various preparations indicates that there is no apparent contribution of this class of compounds in the cytotoxicity of the lipophilic extract (Table [1] and Fig. [3] B).

In conclusion, our data indicate that the antitumor activity observed in bladder cancer cell lines is affected by as yet unidentified lipophilic components of the herb. The lipophilic (PEE) extract of the herb exhibits strong antiproliferative activity, while naphthodianthrones do not contribute to this activity. The same is applicable to the chlorophylls of the herb. Hyperforin, even though a lipophilic compound with documented cytotoxic properties, does not seem to contribute significantly to the antitumor effect of our lipophilic extract. Further investigation is currently under way to identify the chemical component(s) of the PEE, and its active fraction PEE6, which are responsible as separate entities, or which may act in a synergistic manner to cause the significant cytotoxic activity.

Acknowledgements

This work was supported in part by an E.C. grant from General Secretary of Research & Technology of Greece (grant 35-Spin off 2001). The authors would like to thank Professor T. Fotsis, for his advice and support, Dr. Carol Murphy for coordination, and supervision of the in vitro experiments, and Lambrini Kirkou for carrying out the in vitro work in the laboratory of Biological Chemistry (Medical School, University of Ioannina).

References

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Dimitris Skalkos

Department of Material Sciences & Engineering

University of Ioannina

Ioannina GR-45110

Greece

Phone: +32-651-097-262

Fax: +32-651-097-056

Email: dskalkos@cc.uoi.gr

References

• 1 Landis S H, Murray T, Bolden S, Wingo P A. Cancer Statistics, 1998.  CA Cancer J Clin. 1998;  48 6-29
  CrossrefPubMedSearch in Google Scholar
• 2 Koss L G. Tumors of bladder. In: Koss LG, editor Diagnostic Cytology of the Urinary Tract With Histopathology and Clinical Correlations. New York; Lippincott-Raven 1995: pp 71-80
  Search in Google Scholar
• 3 Kurth K H, Boufoux C, Sylvester R, van der Meijden A PM, Oosterlinck W, Brausi M. Treatment of superficial bladder tumors: achievements and needs.  Eur Urol. 2000;  37 1-9
  PubMedSearch in Google Scholar
• 4 Nseyo U O, Lamn D L. Therapy of superficial bladder cancer.  Semin Oncol. 1996;  23 598-604
  PubMedSearch in Google Scholar
• 5 Stavropoulos N E, Hastazeris K, Filiadis I, Michailidis I, Ioachim E, Liamis Z. et al . Intravesical instillations of interferon gamma in the prophylaxis of high risk superficial bladder cancer - results of a controlled prospective study.  Scand J Urol Nephrol. 2002;  36 218-22
  PubMedSearch in Google Scholar
• 6 Prout GR J r, Barton B A, Grifin P P, Friedell G H. Treated history of non-invasive grade 1 transitional cell carcinoma.  J Urol. 1992;  148 1413-9
  PubMedSearch in Google Scholar
• 7 Nseyo U O, DeHaven J, Dougherty T J, Potter W R, Merill D L, Lundahl S L. et al . Photodynamic therapy (PDT) in the treatment of patients with resistant superficial bladder cancer: a long-term experience.  J Clin Laser Med Surg. 1998;  16 61-8
  PubMedSearch in Google Scholar
• 8 Blumenthal M, Busse W R, Goldberg A, Gruenwald J, Hall T, Riggins C W, Rister R S, editors . The complete German Commission E monographs: therapeutic guide to herbal medicines. Boston; American Botanical Council 1998
  Search in Google Scholar
• 9 St John’s W ort. (Hypericum perforatum). In: Upton R editor American herbal pharmacopoeia (AHP). St. Cruz; 1997: pp 1-32
  Search in Google Scholar
• 10 Malamas M, Marselos M. The tradition of medicinal plants in Zagori, Epirus (Northwestern Greece).  J Ethnopharmacol. 1992;  37 197-203
  CrossrefPubMedSearch in Google Scholar
• 11 Brantner A, Della Loggia R, Sosa S, Kartnig T. Untersuchungen zur antiphlogistichen Wirkung von Hypericum perforatum L.  Sci Pharm. 1994;  62 7-8
  PubMedSearch in Google Scholar
• 12 Shakirova K K, Garagulya A D, Khazanovich R L. Antimicrobial properties of some species of St. John’s wort cultivated in Uzbekistan.  Mikrobiol Zh. 1970;  32 494-7
  PubMedSearch in Google Scholar
• 13 Yip L, Hudson J B, Gruszecka-Kowalik , Zalkow L H, Towers G H. Antiviral activity of a derivative of the photosensitive compound hypericin.  Phytomedicine III. 1996;  2 185-90
  PubMedSearch in Google Scholar
• 14 Harrer G, Sommer H. Treatment of mild/moderate depressions with Hypericum .  Phytomedicine. 1994;  1 3-8
  PubMedSearch in Google Scholar
• 15 Linde K, Murlow C J, Berner M, Egger M. St John’s wort for depression.  Cochrane Database Syst Rev. 2005;  18 CD000448
  PubMedSearch in Google Scholar
• 16 Bilia A R, Gallori S, Vincieri F F. St John’s wort and depression efficacy, safety and tolerability - an update.  Life Sci. 2002;  70 3077-96
  CrossrefPubMedSearch in Google Scholar
• 17 Pietta P, Gardana C, Pietta A. Comparative evaluation of St. John’s wort from different Italian regions.  Il Farmaco. 2001;  56 491-6
  CrossrefPubMedSearch in Google Scholar
• 18 Singer A, Wonneman M, Mueller W E. Hyperforin, a major antidepressant constituent of St. John’s wort inhibits serotonin uptake by elevating free intracellular Na^{+ 1} .  J Pharmacol Exp Ther. 1999;  290 1363-8
  PubMedSearch in Google Scholar
• 19 Butterweck V, Petereit F, Winterhoff H, Nahrstedt A. Solubilized-hypericin and pseudohypericin from Hypericum perforatum exert anti-depressant activity in the forced swimming test.  Planta Med. 1998;  64 291-4
  Thieme ConnectPubMedSearch in Google Scholar
• 20 Butterweck V, Jurgenliemk G, Nahrstedt A, Winterhoff H. Flavonoids from Hypericum perforatum show antidepressant activity in the forced swimming test.  Planta Med. 2000;  66 3-6
  Thieme ConnectPubMedSearch in Google Scholar
• 21 Skalkos D, Tatsis E, Gerothanasis I P, Troganis A. Towards a consensus structure of hypericin in solution: direct evidence for a single tautomer and different ionization states in protic and nonprotic solvents by the use of variable temperature gradient ¹H NMR.  Tetrahedron. 2002;  58 4925-9
  CrossrefPubMedSearch in Google Scholar
• 22 United States P harmacopoeia. St. John’s wort monograph, 9^th Supplement. 1998: pp 4709-4710
  Search in Google Scholar
• 23 AOAC method 9 42.04, Chlorophylls in p lants. In: Cunniff P, editor Official Methods of Analysis of AOAC International. Gaithersburg, MD; 1998
  Search in Google Scholar
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Dimitris Skalkos

Department of Material Sciences & Engineering

University of Ioannina

Ioannina GR-45110

Greece

Phone: +32-651-097-262

Fax: +32-651-097-056

Email: dskalkos@cc.uoi.gr

• Supplementary Material