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Antitumor and immunostimulating effects of Anoectochilus formosanus Hayata.

Abstract

The water extract of Anoectochilus formosanus Hayata showed a potent tumor inhibitory activity in BALB/c mice after subcutaneous transplantation of CT-26 murine colon cancer cells. The tumor-inhibition ratios of mice pre-administered with A. formosanus for 2 days before tumor transplantation, and treated further for 12 consecutive days, were 55.4% and 58.9% at the oral dose of 50 and 10 mg/mouse per day, respectively. Even for the tumor-bearing mice, after oral administration of the water extract of A. formosanus for 12 consecutive days, the tumor inhibition ratios were still 23.8% and 40.5% at doses of 50 and 10 mg/mouse, respectively. Because the low-concentration water extract of A. formosanus does not show direct cytotoxicity in CT-26 tumor cells, we observed further that oral administration of the water extract of A. formosanus may activate murine immune responses, such as stimulating the proliferation of lymphoid tissues and activating the phagocytosis of peritoneal macrophages against Staphylococcus aureus. This study suggests that the antitumor activity of A. formosanus may be associated with its potent immunostimulating effect. It is worth further analyzing the immunomodulating component purified from A. formosanus, and evaluating its potential value for the treatment of human cancers.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Anoectochilus formosanus; In vivo antitumoral and immunostimulating activity

Introduction

The genus Anoectochilus (Orchidaceae), comprising more than 35 species in the world, is a perennial herb. It is distributed from India, the Himalayas, Southeast Asia, and Indonesia to New Caledonia and Hawaii (Hsu, 1978). Of the species, four are found in Taiwan, including Anoectochilus formosanus, Anoectochilus inabai, Anoectochilus koshunensis, and Anoectochilus lanceolatus. A. formosanus and A. koshunensis have been used by local people to cure snake bite. Anoectochilus spp. are now expensive folk medicinal plants used to treat cancer, hypertension, diabetes mellitus, consumption, and nephitis in Taiwan (Liang et al., 1990). A. formosanus Hayata has been called "King Medicine", because of its diverse pharmacological effects, including anti-inflammation and hepatoprotective activities (Lin et al., 1993), and antioxidant activities (Lin et al., 2000; Wang et al., 2002). However, there is still no report on the antitumor or immunomodulatory activity of A. formosanus. Because some plant polysaccharides are well known to possess immunoregulatory effects and to exhibit significant antitumor activities (Roesler et al., 1991; Wong et al., 1994), and A. formosanus has been a popular folk medicine for treating cancer in Taiwan, the purpose of this study was to evaluate the antitumor activity of A. formosanus in mice and to explore its mode of action by studying its immunomodulating effect. In order to investigate its immunomodulating activity, the proliferative responses of lymphocytes to both T- and B-cell mitogens, and phagocytosis activity against Staphylococcus aureus infection were examined.

Materials and methods

Animals

Female BALB/c mice (Animal Center, Taiwan University, Taiwan) weighing 16-18 g each were used for all experiments. Animals received a standard laboratory chow diet and water ad libitum, prior to the studies.

Preparation of water extract of A. formosanus

A. formosanus Hayata was collected from Puli in central Taiwan. The species of the plant was authenticated and a voucher specimen (Accession No. SP 9703010) was deposited in the herbarium of Taipei Medical University (Cheng et al., 1998). To obtain the water extract of A. formosanus, four volumes (v/w) of cold water were added to the whole A. formosanus plant and homogenized in the blender. After filtration though cheesecloth, the filtrate was further centrifuged at 12,000 rpm for 1 h at 4[degrees]C. The supernatant was freeze-dried by lyophilization.

Antitumor activity of A. formosanus

BALB/c mice were orally administered with various concentrations of water extract of A. formosanus for 2 days before subcutaneous implantation with CT-26 murine colon cancer cells ([10.sup.6] cells/mouse). We treated the tumor inoculated mice continuously with the same dose of A. formosanus for 12 consecutive days. In order to evaluate its antitumor therapeutic effect, after the tumors reached a size of approximately 0.5 [cm.sup.3] (8 days after CT-26 cell inoculation), the tumor-bearing mice received various concentrations of water extract of A. formosanus orally, while the control group received water only for 12 consecutive days. The antitumor activity of A. formosanus was determined by measuring the tumor weight on day 7 after ceasing treatment.

Phagocytosis of murine peritoneal macrophages

Peritoneal exudate macrophages were harvested from BALB/c mice 3 days after intraperitoneal injection of 2 ml of thioglycollate liquid medium (Difco) (Kuwano et al., 1993). The macrophages were cultured in 96-well, flat-bottom tissue culture plates (Falcon, Becton Dickinson, Co., Oxford, CA), and each well contained 1 x [10.sup.5] peritoneal cells in 200 [micro]l of RPMI 1640 supplemented with 5% fetal calf scrum (FCS). After 2 h incubation at 37[degrees]C in a 5% C[O.sub.2] incubator, the macrophages were washed vigorously three times with warmed Hanks' balanced salt solution (Nissui Selyaku Co., Tokyo) containing 0.1% gelatin (Difco Lab.) and then fed with 100 [micro]l RPMI/5% FCS. The recovered macrophages in the crude cell preparations had 70-80% purity, as revealed by myeloperoxidase staining. Endotoxin in the medium was less than 1 ng/ml by the Limulus test (Endotoxin test-D; Seikagaku Kogyo Co., Ltd., Tokyo, Japan). The attached macrophages were treated with various concentrations of A. formosanus for 24 h at 37[degrees]C, and we then investigated their phagocytosis activity against S. aureus (ATCC 25923) and MTT reduction activity. The cell ratio between macrophages and added bacteria was 1:25. After 4h incubation with bacteria, the phagocytosis activity was measured by counting the extracellular bacteria numbers on Mannitol salt agar plate (Difco Lab.), a selective medium for S. aureus.

Mitogen-stimulation assay

The murine splenocytes were used to assess lymphocyte proliferation response after mitogen stimulating for 20h. Phytohemagglutinin (PHA, Siga) was used as T-lymphocyte mitogen and lipopolysaccharide (LPS, Escherichia coli 055:B5, Siga) as a B-lymphocyte mitogen. The optimal stimulating concentrations were 50 and 10 [micro]g/ml, respectively (Puri et al., 1996).

Assay for MTT activity (Garn et al., 1994; Ferrari et al., 1990)

After mitogen stimulation of the spleen cells, 20 [micro]l/well of tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide, Siga, St. Louis) solution (5 mg/ml in RPMI/5% FCS) were added, and the plates were incubated at 37[degrees]C in 5% C[O.sub.2]-balanced air for another 4h. The supernatant of untransformed MTT was aspirated carefully after centrifuging the tray. The reduced tetrazolium precipitates were solubilized in 200 [micro]l/well DMSO (Siga, St. Louis) with vigorous mixing in a microplate shaker for a few minutes. Thereafter, the optical densities were read by an automatic plate reader (Bio Tek Instrument Winooski, USA) at a wavelength of 540 nm.

Statistical analysis

Differences between groups were evaluated for significance using the Student's t-test and the Kruskal--Wallis test. A p value of < 0.05 was considered significant.

Results

Long tent toxicity of A. formosanus in BALB/c mice

After oral administration of the water extract of A. formosanus for 42 consecutive days, at a dose of 100 mg/mouse, the treated BALB/c mice exhibited no obvious change in their body weight, glistening of hair, physical activity or biological function of the liver (the concentration of GPT, data not shown).

Antitumor activity of the water extract of A. formosanus against subcutaneously transplanted CT-26 murine colon cancer cells in BALB/c mice

The antitumor activity of A. formosanus is shown in Table 1. The BALB/c mice were administered the water extract of A. formosanus orally for 2 days before tumor cell inoculation, and then were treated continuously with the same dose of A. formosanus for 12 consecutive days. Tumor weights of A. formosanus-treated groups were 0.25[+ or -]0.13 and 0.23[+ or -]0.10 g for the doses of 50 and 10 mg/mouse, respectively, while the tumor weight of the control group was 0.56[+ or -]0.12 g. Therefore, the water extract of A. formosanus showed excellent inhibitory activity against solid tumor formation of CT-26, with inhibitory ratios of 55.4% and 58.9% for the 50 and 10 mg/mouse groups, respectively. The therapeutic tumor inhibitory activity of A. formosanus for the tumor-bearing mice was also investigated and results are shown in Table 2. After the tumor size of BALB/c mice reached approximately 0.5 [cm.sup.3], treatment was initiated with A. formosanus for 12 consecutive days. The tumor weights of the 50 and 10 mg/mouse groups were 0.32[+ or -]0.13 and 0.25[+ or -]0.17 g, respectively, vs. 0.42[+ or -]0.16 g for the control group. Therefore, the water extract of A. formosanus showed significant tumor inhibitory activity in tumor bearing-mice.

Stimulating effects of A. formosanus on murine peritoneal macrophages

As shown in Table 3, the stimulating effects of A. formosanus on murine peritoneal macrophages were investigated by measuring the phagocytic activities in vitro as well as MTT reduction activity. Comparing to the control group, the extracellular bacterial colony forming unit (CFU) counts for the group treated with 0.05 mg/ml A. formosanus decreased significantly (p<0.05), whereas the group treated with 5.0 mg/ml showed strong suppression of phagocytic activity against S. aureus. We also investigated the effects of the water extract of A. formosanus on MTT activity in macrophages. The 0.05 mg/ml-dose macrophages showed enhanced MTT activity, whereas the 5.0mg/ml-dose group showed strong inhibition of MTT reduction activity. Because the MTT reduction activity represents the viability of macrophages, macrophages treated with 5.0 mg/ml A. formosanus showed only 0.02[+ or -]0.01 MTT activity, meaning that a 5.0 mg/ml dose of A. formosanus might be cytotoxic to the murine peritoneal macrophages. This result could explain why the proper concentration of A. formosanus for stimulating the activity of murine peritoneal macrophages was 0.05 mg/ml, instead of 0.5 or 5.0 mg/ml. Therefore, the in vivo antitumor activity of A. formosanus might be due to its immunomodulating effects, but not its cytotoxicity on tumor cells directly.

The proliferation response of splenic lymphocytes after mitogen stimulation

In this study, we used the MTT assay to detect the proliferation activity of splenocytes after T-lymphocyte (PHA, 10 [micro]g/ml) and B-lymphocyte mitogen (LPS, 50 [micro]g/ml) stimulation. As shown in Table 4, the splenocyte viability of A. formosanus-pretreated mice was better than that of control mice (0.95[+ or -]0.15, 1.00[+ or -]0.25 vs. 0.78[+ or -]0.18). After mitogen stimulation, both the T- and B-lymphocytes in the spleen of A. formosanus-pretreated mice showed stronger proliferation activity. Mice which received 10 mg/mouse of A. formosanus pretreatment, showed significant splenocyte-stimulating effects by mitogens PHA and LPS (p<0.001) as compared to the control.

Discussion

A. formosanus is a popular folk medicine that is used as a remedy for hepatitis, hypertension, tuberculosis and cancer in Taiwan (Hsu, 1978; Liang et al., 1990). We have shown that long-term administration of the water extract of A. formosanus had no detectable harmful effect on the health of BALB/c mice. Lin et al., (2000) reported that the methanol extract of A. formosanus exhibited antilipid peroxidation, antisuperoxide formation and free-radical-scavenging activity. Another study indicated that the water extract of A. formosanus might completely prevent induced oxidative stress with [H.sub.2][O.sub.2] or xanthine treatment in HL-60 cells. These reports did not show that A. formosanus might have any antitumor effect, although A. formosanus has long been used as a folk medicine to treat cancer in Taiwan.

In this study, we found that oral administration of A. formosanus could significantly prevent and inhibit CT-26 solid tumor formation in BALB/c mice. In our investigation to understand the mechanism of its antitumor activity, we found that the water extract of A. formosanus could kill the CT-26 tumor cells in vitro at high concentration (5.0 mg/ml) though stimulating the apoptosis processes of tumor cells, but that its cytotoxicity to CT-26 tumor cells at 0.5 mg/ml is only 27.6% (data not shown). According to the published reports, some well-known crude drugs used in traditional Chinese medicine or some natural plant extracts' multiple biological activities in wound healing, anti-inflammation or immunomodulation are due to their water-soluble polysaccharides (Roesler et al., 1991; Wong et al., 1994; Strickland et al., 1999; Zhihua et al., 2000). A set of polysaccharides with different molecular weights has been isolated by gel fraction and identified as enhancing the immune responses, especially stimulating macrophage activities (Veronique and Bang, 1999). Macrophages are the antigen-presenting cells known to be both cytotoxic and phagocytic to tumor cells and invading microorganisms. They play a very important role in linking our innate and adaptive immunities, so that these two types of immunity can act cooperatively and interdependently (Engelhard, 1994; Fearon and Locksley, 1996). Therefore, stimulation of macrophages might be a major target for therapeutic application (Compton et al., 1996; Zhihua et al., 2000). In this study, we found that the water extract of A. formosanus could enhance the viability and phagocytosis of murine peritoneal macrophages at low concentration. In addition, we also found that the water extract of A. formosanus could increase the proliferation of murine splenocytes after stimulation by either T or B mitogen. We plan to further analyze the cytokines secretion of these immune cells, such as TNF-[alpha], IL-1, IL-2, IL-4, etc., which may play a critical role in stimulating the immune defense system against tumor cells. In summary, this study demonstrated that the water extract of A. formosanus might not only be used as a hepatoprotective drug but also as a prominent immunomodulating agent. It will be worth further purifying the potent immunomodulating components from A. formosanus, and evaluating the plant's potential value in the treatment of human cancers.

Acknowledgements

The study was supported by a financial grant from the National Science Council of the Republic of China (NSC 90-2317-B-038-002).

References

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C.-C. Tseng (a), H.-F. Shang (a), L.-F. Wang (b), B. Su (b), C.-C. Hsu (c), H.-Y. Kao (a), K.-T. Cheng (b,*)

(a) Department of Microbiology and Immunology, School of Medicine, Taipei Medical University, Taipei, Taiwan

(b) Department of Biochemistry, School of Medicine, Taipei Medical University, Taipei, Taiwan

(c) Department of Anatomy, School of Medicine, Taipei Medical University, Taipei, Taiwan

*Corresponding author. Tel.: +886 02 27361661 3169: fax: +886 02 27399650.

E-mail address: ktbot@tmu.edu.tw (K.-T. Cheng).
Table 1. Antitumor activity of the water extract of A. formosamus
against CT-26 tumor cells in BALB/c mice

AF concentration Tumor weight (g)
(mg/mouse) (mean [+ or -] s.e.m.) % Inhibition

50 0.25 [+ or -] 0.13* 55.4
10 0.23 [+ or -] 0.10* 58.9
 0 0.56 [+ or -] 0.12 0

In all, 1 x [10.sup.6] CT-26 murine colon caneer cells/mouse were
subcutaneously injected in BALB/c mice which had been orally
administrated with 50 or 10 mg/mouse/day A. formosanus water extract
(AF) for 2 days. After tumor implantation, the mice were continuously
administered the same dose of A. formosanus continuously for 12
consecutive days. The wet weight of the tumor mass was measured on day 7
after ceasing treatment. Each group consisted of ten BALB/c mice.
*Statistically significant difference from the control group of zero
concentration as measured by the Student t-test. *p < 0.01.

Table 2. Tumor-inhibitory effect of the water extract of A. formosanus
in CT-26 tumor-bearing BALB/c mice

AF treatment Tumor
(mg/mouse) weight (g) Inhibition (%)

50 0.32 [+ or -] 0.13 23.8
10 0.25 [+ or -] 0.17* 40.5
 0 0.42 [+ or -] 0.16 0

In all, 1 x [10.sup.6] CT-26 murine colon cancer cells/mouse were
subcutaneously injected in BALB/c mice. When the tumor size reached
approximately 0.5 [cm.sup.3], the mice were split into three groups of
ten BALB/c mice each. Various concentrations of the water extract of A.
formosanus (AF) were administered to the tumor-bearing mice orally for
12 consecutive days. Tumor weight was measured on day 7 after ceasing
treatment.
*Statistically significant difference from zero concentration treated
group as measured by the Student t-test, *p<0.05.

Table 3. Phagocytosis against S. aureus and MTT activity of murine
peritoneal macrophages stimulated by the water extract of A. formosanus

AF treatment Extracellular bacteria (a) MTT activity (b)
(mg/ml) (x [10.sup.6] CFU/ml) (OD 540nm)

0 31 [+ or -] 4.5 (31) 0.16 [+ or -] 0.07
0.05 17 [+ or -] 2.6 (16)* 0.32 [+ or -] 0.12*
0.5 46 [+ or -] 18.4 (54) 0.19 [+ or -] 0.06
5.0 456 [+ or -] 50.3 (438) 0.02 [+ or -] 0.01*

In all, 1 x [10.sup.5] cells per well of peritoneal macrophages in
100 [micro]l of RPMI/FCS were pretreated with various concentrations of
the water extract of A. formosanus (AF) at 37[degrees]C for 24 h,
followed by adding of S. aureus suspension (1:25) or MTT
(100 [micro]g/well). After 4 h incubation, the phagocytosis and MTT
reduction activities were determined as described in Materials and
methods.
(a) Statistically significant difference from zero-concentration group
as measured by the Kruskal-Wallis test, *p<0.05. Numbers in
parentheses show the median number of bacterial CFU in each group.
(b) Statistically significant difference from zero control group as
measured by the Student t-test *p<0.05. Each group consisted of 12-24
wells in a 96-well cultural microplate.

Table 4. Mitogen-stimulating effects on the splenic lymphocytes of A.
formosanus-pretreated mice

 (OD 540 nm)
AF treated MTT reduction activity LPS
(mg/mouse) Control PHA (10 [micro]g/ml) (50 [micro]g/ml)

50 0.95 [+ or -] 0.15* 0.95 [+ or -] 0.18 1.02 [+ or -]
 0.17**
10 1.00 [+ or -] 0.25* 1.33 [+ or -] 0.25*** 1.23 [+ or -]
 0.19***
 0 0.78 [+ or -] 0.18 0.88 [+ or -] 0.12 0.73 [+ or -]
 0.19

The results were expressed as mean [+ or -] s.e.m. MTT activity was used
to detect the proliferation response of splenic lymphocytes after
mitogen stimulation. Before the splenocyte harvest, tested animals were
administered the water extract of A. formosanus (AF) orally for 12
consecutive days. Each group consisted of seven BALB/c mice.
Statistically significant differences from zero group as measured by
the student t-test: *p < 0.005, **p < 0.01 and ***p < 0.001.
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Author:Tseng, C.-C.; Shang, H.-F.; Wang, L.-F.; Su, B.; Hsu, C.-C.; Kao, H.-Y.; Cheng, K.-T.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:May 1, 2006
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