Stephania venosa-edited-DONE_2nd

Original Article

Cytotoxic Effects of Water Extract from Stephania venosa Tubers Tada Sueblinvong1, Tipsuda Plumchai2, Pathama Leewanich3 and Wacharee Limpanasithikul2* 1

Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Department of Pharmacology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand 3 Department of Pharmacology, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110, Thailand * Corresponding author: [email protected] 2

ABSTRACT Stephania venosa (Bl.) Spreng (S. venosa) is a Thai medicinal plant locally known as “Saboo luad” or “Boraphet plungchang”. It was used for treating various illnesses including cancer. This study aimed to investigate cytotoxic activity of the water extract from S. venosa tubers on human peripheral blood mononuclear cells (PBMCs). PBMCs from healthy subjects were used to examine cytotoxic effect of the extract by using trypan blue dye exclusion method. The results demonstrated cytotoxic activity of the extract with its IC50 at 300 mg/mL. The effect of the extract on apoptotic induction was also evaluated at the concentration of 300 mg/mL on PMBCs from healthy subjects and from cervical cancer patients by using the terminal deoxynucleotidyl transferase (TUNEL) assay. The results revealed that the extract significantly induced apoptosis of the PBMCs from both healthy subjects and from the patients, similar to 60Co radiation at 0.5 Gy, when compared to the untreated cells. The additive effect on apoptosis induction was also observed when the extract was used in combination with 60Co radiation to treat the PBMCs from healthy subjects. We also studied the antiproliferative effect of the extract on the cells from healthy subjects by using by 3H-thymidine incorporation assay. It was demonstrated that the extract had antiproliferative activity with IC50 at 40 mg/ mL. Taken together, these results suggest that the water extract of S. venosa tubers possess antitumor potential via cytotoxic, apoptotic and anti-proliferative properties. Key Words: Stephania venosa, cytotoxicity, apoptosis, anti-proliferation §

Thai Pharm Health Sci J 2007;2(3):203-208

Introduction Stephania venosa (Bl.) Spreng is a plant in the family of Menispermaceae. Tubers of this plant have been used in Thai traditional medicine for nerve tonic, aphrodisiac, appetizer, and for treatment of asthma, microbial infection, hyperglycemia and cancer. Over thirty isoquinoline alkaloids in the tubers have been identified and demonstrated many pharmacological activities, as well as cytotoxic potential.1-9 The water and ethanol extracts from the tuber of S. venosa showed cytotoxic activity against brine shrimps with the IC50 at 184.9 and §

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12th year of Srinakharinwirot Journal of Pharmaceutical Science

90.8 µg/mL, respectivel.6 The ethanol extract exhibited cytotoxic activity against human breast cancer cell line, MCF-7, with the IC50 at 11.6 µg/mL.9 However, it is not known about the molecular and cellular mechanism responsible for this cytotoxic effect on the cancer cells. It is known that various anticancer agents can eliminate cancer cells through various activities, i.e., cytotoxicity, inhibition of tumor cell proliferation, inhibition of angiogenesis, or activation of programmed cell death or apoptosis. In the present study, we examined cellular effects of the water extract of S. venosa tubers toward human peripheral blood mononuclear cells (PBMCs) from

Thai Pharmaceutical and Health Science Journal, Vol. 2 No. 3, Sep. – Dec. 2007

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healthy subjects and from patients with cervical cancer. The cytotoxic, antiproliferative and apoptogenic activities of the extract were evaluated.

Materials and Methods Plant material and extract preparation

signed their inform consent forms and the study project had been approved by The Ethics Committee of the Faculty of Medicine, Chulalongkorn University. Six patients were diagnosed as staged IIB and the other three had stage IIIB cervical cancer; they were 41 - 76 years old. Patients who were HIV positive or had either been through radiotherapy or chemotherapy were excluded.

Authentication of the tuber of S. venosa (Bl.) Spreng was achieved by comparison with herbarium specimens at the Bangkok Forest Herbarium, Royal Forest Department, Ministry of Agriculture and Cooperatives, Bangkok, Thailand. S. venosa tubers were collected from a forest in Rachaburi province, Thailand. The fresh tubers were harvested, chopped into small pieces and dried. Thirty grams of the dried tubers in 2 liters of distilled water was extracted by decoction for 5 h. The solution was filtered by filter paper and lyophilized. The dried extract was stored in a desiccator at 4 °C until use.

The cytotoxic effect of water extract on PBMCs was determined by trypan blue dye exclusion method. Human PBMCs (1x105 cells/well) were treated with the extract at various concentrations (18 - 600 mg/mL) in a 96-well plate for 48 h before staining with 0.4% trypan blue solution. The cytotoxic effect was evaluated by comparing with untreated condition. Actinomycin D at the concentration of 0.08 mM was used as the positive control. All experiments were performed in triplication.

Reagents, culture medium, and cell preparation

Apoptosis determination

The following chemicals and reagents were purchased from Sigma (USA): streptomycin and penicillin, L-glutamine, HEPES and Hoeschts 33258 dye solution. Fetal bovine serum, Hanks’ Balanced Salts Solution (HBSS) Powder and L-glutamine RPMI 1640 medium were purchased from Gibco (USA). FicollHypaque was purchased from Amersham (USA). ApopTag® Fluorescein kit was from Intergen (USA). To prepare human PBMCs, heparinized blood obtained from healthy female donors, who attended the National Blood Bank, Thai Red Cross Society for blood donation, were centrifuged using Ficoll-Hypaque. Cells were maintained in RPMI 1640 containing 10% fetal bovine serum, 20 mM HEPES (pH 7.0), 2 mM Lglutamine, and 1% penicillin-streptomycin. PBMCs were also isolated from heparinized blood of cervical cancer patients who attended Cervical Cancer Clinic at the Division of Radiotherapy, Department of Radiology, King Chulalongkorn Memorial Hospital by Ficoll-Hypaque gradient centrifugation. Nine patients had 204

Cytotoxic assay

The apoptogenic effect of the extracts was determined by using the TUNEL assay. Human PBMCs (4 x 105 cells/mL) were treated with the extracts at the concentration of 300 mg/mL for 48 h and then were pelleted down by refrigerated centrifugation at 2,000 rpm (4oC). The treated cells were proceeded on slides for the detection of apoptosis by ApopTagâIn situ apoptosis detection kit (TdT assay) according to the manufacturer’s protocol. The apoptotic cells were evaluated under a fluorescence microscope. The apoptotic index of the extract was then calculated according to the following equations: Apoptotic index = Number of apoptotic cells x 100 Number of total cells

..... (1)

Apoptotic activity = Apoptotic index T – Apoptotic indexU …. (2)

Note: Apoptotic index T = Apoptotic index of the treated sample Apoptotic indexU = Apoptotic index of the untreated control



The experiments were performed in triplication. 60Co exposed PBMCs, at 0.5 Gy, was used as positive condition. The combined effect between the extract and the 60Co radiation on apoptotic induction was also examined.

Proliferation assay

was similar to 0.5 Gy 60Co radiation which caused apoptosis at 22.2 ± 1.81 %. The additive effect of the combination between the extract and the radiation was also observed in both conditions, the extract prior to radiation and the radiation prior to the extract treated conditions (Figure 2).

The inhibitory effect of the extract was determined by using 3H–thymidine incorporation assay. Human PBMCs (2x105 cells/well) were stimulated with 0.01 mg/ mL phytohemagglutinin (PHA) in the presence of various concentrations of the extract (3 - 1,000 mg/mL) in 96-well plate for 24 h. 3H–Thymidine at 1 mCi was added in each well and the cells were further incubated for 6 h before harvested and counted by scintillation counter. All experiments were performed in triplication.

Statistical analysis

All data were presented as means and standard error of means (mean ± SEM). Repeated measurement test was used to compare the significance between the control and treatment groups. A p-value less than 0.05 was set for the level of significant difference.

Figure 1 The cytotoxic effect of the water extract from S. venosa tubers on healthy human PBMCs. Data expressed as the mean percentage of untreated cells ± SEM (n = 5).

Results Cytotoxic activity of the water extract of S. venosa The water extract of S. venosa tuber significantly decreased healthy human PBMC viability in a dose dependent manner by trypan blue dye exclusion assays with IC50 at 300 mg/mL (Figure 1).

Figure 2 Apoptotic index of the extract against healthy

Apoptotic activity of the water extract of S. venosa on PBMCs The water extract at the concentration of 300 mg/ mL was significantly induced apoptosis on PBMCs from healthy subjects (21.8 ± 1.29% apoptosis) when compared to the untreated control (6.3 ± 0.37%) (Figure 2). The apoptogenic activity of the 300 mg/mL extract

human PBMCs. The cells were treated with 300 mg/mL S. venosa, 0.5 Gy 60Co radiation, combination I (S. venosa prior to radiation) or combination II (radiation prior to S. venosa) for 48 h. Data expressed as mean ± SEM (n = 10). *P < 0.05 different from the untreated control; #P < 0.05 different from S. venosa or radiation alone.



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When the PBMCs from patients with cervical cancer were used to investigate the apoptogenic activity of the extract, the apoptotic index of the extract at 300 (g/mL (28.6 ± 3.92 %) was significantly higher than that in the untreated control (10.0 ± 1.75 %) but similar to that from radiation (29.2 ± 3.00 %) (Figure 3). No additive effect was demonstrated when the extract was combined with the radiation for apoptotic induction on the cells from the patients. The apoptotic activity of the extract on PBMCs from these patients was not significantly different from the PBMCs of the healthy subjects.

A p op totic in de x (% )

50 40 *

30

*

*

20

Discussion

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C on trol

S . ve no sa

R ad iatio n

R ad iation + S . ve no sa

Figure 3 Apoptotic index of the extract against PBMCs from cervical cancer patients stage IIB & IIIB. The cells were treated with 300 mg/ mL S. venosa, 0.5 Gy 60Co radiation or the combination of the extract and radiation for 48 h. Data expressed as mean ± SEM (n = 9). * Apoptotic index significantly different from the untreated control at P < 0.05.

Antiproliferative effect of the water extract of S. venosa The effect of the water extract of S. venosa tuber on PHA-stimulated healthy human lymphocyte proliferation was determined by [3H] thymidine incorporation. The result showed that S. venosa inhibited lymphocyte proliferation in a dose dependent pattern with IC50 at 40 mg/mL (Figure 4).

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Figure 4 Antiproliferative effect of the extract against healthy human PBMCs. The PHA-stimulated PBMCs were treated with various concentrations of S. venosa. Data expressed as the mean percentage of inhibition of proliferation ± SEM (n = 6).

Stephania venosa is one of Thai traditional medicines used for cancer treatment. The cytotoxic effect of S. venosa tubers had been reported against brine shrimps and human breast cancer cells.6,9 In this study, the cellular mechanisms responsible for antitumor potential of the water extract of S. venosa tubers were evaluated on human PBMCs. The extract demonstrated the cytotoxic activity in a dose dependent manner. It was reported that palmatine and crebanine, isolated from the ethanol extract of S. venosa, were mainly responsible for the cytotoxic activities.9 Moreover, palmatine was recently found to bind strongly to single stranded poly(A) molecules.10 Using gas chromatography-mass spectrometry techniques (GC-MS), we found that an isoquinoline alkaloid domestine was the main compound from the water extract of S. venosa (unpublished data) but its biological activity was rarely identified. Tetrahydropalmatine is another compound found in both water and ethanol extracts of S. venosa but it is less potent on cytotoxicity than palmatine.9 Thus, to identify the main compound responsible for the cytotoxic activity of the water extract, domestine is an interest.



Cell death induced by anticancer agents has been considered to be a consequence of a block in proliferation. It is known that many anticancer drugs can inhibit cell proliferation by blocking DNA synthesis of cancer cells as well as rapid dividing cells. Proliferation of PHA-stimulated human PBMCs was used to evaluate antiproliferative activity of the water extract of S. venosa tubers in this study. The results showed that the extract had more potent antiproliferative effect than its cytotoxic effect, with IC50 40 mg/mL and 300 mg/mL, respectively. This suggests that inhibition of cell proliferation may be one of the cellular mechanisms of the anti-tumor activity of S. venosa tubers. Apoptotic induction is another cellular mechanism of anticancer drugs. Over the past decade, studies have shown that many anticancer agents can induce apoptosis and their molecular mechanisms have been clarified.11-16 The water extract of S. venosa tubers was also demonstrated apoptotic induction on PBMCs in this study. The potency of this activity was similar to that of 60 Co radiation but less than anti-proliferative activity. So, apoptotic induction may be one of the cellular mechanism of S. venosa tubers that cause cell death. In summary, the water extract of S. venosa tubers has anti-tumor potential by exhibiting cytotoxic, antiproliferative, and apoptotic activities on human PBMCs. These anti-tumor activities on cancer cells should be further investigated for the risk and the benefit of the tubers for cancer treatment.

Acknowledgements

This work was supported by Thailand Government through Srinakharinwirot University, research grants from the Ministry of University Affairs through Chulalongkorn University, and by the Chulalongkorn University Graduate School Thesis Grant.

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