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Anticancer activity of the Uncaria tomentosa (Willd.) DC. preparations with different oxindole alkaloid composition.

ABSTRACT

The activity of Uncaria tomentosa preparations on cancer cells was studied using in vitro and in vivo models. [IC.sub.50] values were calculated for preparations with different quantitative and qualitative oxindole alkaloid composition: B/W37 - bark extracted in water at 37 [degrees]C, B/[W.sub.b] - bark extracted in boiling water, B/50[E.sub.37] - bark extracted in 50% ethanol at 37 [degrees]C, B/[E.sub.b] - bark extracted in boiling 96% ethanol, B/96[E.sub.73] - bark extracted in 96% ethanol at 37 C and B/SRT - bark extracted in water and dichloromethane. Generally, the results obtained showed a high correlation between the total oxindole alkaloid content (from 0.43% to 50.40% d.m.) and the antiproliferative activity of the preparations ([IC.sub.50] from > 1000 [micro]g/ml to 23.57 [micro]g/ml). B/96E37 and B/SRT were the most cytotoxic preparations, whereas the lowest toxicity was observed for B/W37. B/96E37 were shown to be active against Lewis lung carcinoma (LL/2) [[IC.sub50] = 25.06 [micro]g/ml], cervical carcinoma (KB) [IC50 = 35.69 [micro]g/ml] and colon adenocarcinoma (SW707) [[IC.sub.50] = 49.06 [micro]g/ml]. B/SRT was especially effective in inhibiting proliferation of cervical carcinoma (KB) [[IC.sub.50] = 23.57 [micro]g/ml], breast carcinoma (MCF-7) [[IC.sub.50] = 29.86 [micro]g/ml] and lung carcinoma (A-549) [[IC.sub.50] = 40.03 [micro]g/ml]. Further animal studies on mice bearing Lewis lung carcinoma showed significant inhibition of tumor growth by B/W37 administered for 21 days at daily doses of 5 and 0.5 mg (p = 0.0009). There were no significant changes in the cell cycles of tumor cells with the exception of cell decrease at the [G.sub.2]/M phase after the administration of B/96[E.sub.37] at a daily dose of 0.5 mg and the [G.sub.1]/[G.sub.0] cells cycle arrest demonstrated after the B/SRT therapy at a daily-dose of 0.05 mg. All tested preparations were non-toxic and well tolerated.

ARTICLE INFO

Keywords: Uncaria tomentosa Una de gato Cat's claw Oxindole alkaloids Anticancer activity

[C] 2010 Elsevier GmbH. All rights reserved.

Introduction

Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle is a woody climbing vine belonging to the Rubiaceae family that grows in the highlands of the Amazon rain forest (Cisneros et al. 2005). Due to its curved hooks this plant is commonly known under the Spanish name 'una de gato' that is translated to English as 'cat's claw' (Keplinger et al. 1999).

Since ancient times, the indigenous people of Peru have used its inner bark and root to prepare medical decoctions (Aguilar et al. 2002; Akesson et al. 2003a; Cisneros et al. 2005; Keplinger et al. 1999). Recently, cat's claw has become more popular and increasingly distributed all over the world as an immunomodulatory, anticancer and anti-inflammatory herbal remedy (De Jong et al. 1999). The list of treated diseases includes gastric ulcers, diarrhoea, gonorrhoea, arthritis and rheumatism, acne, diseases of the urinary tract and cancers (Heitzman et al. 2005).

A number of chemical studies on U. tomentosa revealed the presence of diverse compounds such as alkaloids, quinovic acid glycosides, polyhydroxylated triterpenes, flavonoids, catechins and sterols (Aquino et al. 1989, 1991; De Matta et al. 1976; Heitzman et al. 2005; Kitajima et al. 2004; Muhammad et al. 2001). Wagner and co-workers, already in 1985, demonstrated that some of these compounds (i.e. four oxindole alkaloids: isopteropodine, pteropodine, isomitraphylline, and isorynchophylline) potentiated the phagocytosis by white blood cells (Lemaireetal. 1999; Wagner et al. 1985). Further in vitro and in vivo investigations undertaken by other researchers showed a very complex and multilateral activity of the active constituents of U. tomentosa (Gulewicz et al. 2004; Heitzman et al. 2005; Kuras et al. 2006; Okuhama et al. 2001; Sandoval et al. 2002).

U. tomentosa preparations have been found to show various anti-inflammatory effects such as inhibiting the production of the inflammatory cytokine TNF[alpha] and the activation of the nuclear transcription factor NF-[kappa]B (Akesson et al. 2003a; Miller et al. 1999; Sandoval-Chacon et al. 1998; Sandoval et al. 2000). In other studies, U. tomentosa extracts induced the production of factors that regulate lymphocyte-proliferation by human endothelial cells and the synthesis of IL-1 and IL-6 by rat alveolar macrophages (Lemaire et al. 1999; Wurm et al. 1998). Proapoptotic and antiproliferative effects of aqueous extracts on human myeloid leukaemia cells such as K562 and HL60 have also been described (Sheng et al. 1998). U. tomentosa constituents exerted cytotoxic activity on lymphoblastic leukaemia and breast cancer cells (Bacher et al. 2006; Riva et al. 2001). Furthermore, it has been shown that U. tomentosa extracts enhance DNA repair and protect lymphocytes from apoptosis induced by hydrogen peroxide, diphenyl-2-picrylhydrazyl, and peroxynitrite (Akesson et al. 2005; Sheng et al. 1998). Significant antioxidative properties were also observed in our previous analyses including trolox equivalent antioxidant capacity (TEAC), peroxyl radical-trapping capacity (PRTC) and superoxide radical scavenging activity (SOD) (Pilarski et al. 2006). Aqueous extract was demonstrated to increase the number of leukocytes in normal rats and splenic lymhocytes subsets CD4+, CD8+ and B cells in mice (Akesson et al. 2005; Eberlin et al. 2005). This increase in cell viability and DNA stability could be important for the recovery process of cancer patients after myelosuppression caused by chemotherapy (Allen-Hall et al. 2007; Steinberg 1995).

One of the main problems hindering systematic research on biological activities of U. tomentosa preparations is the different composition of extracts, which makes it difficult to draw direct comparisons between studies. The aim of our study was to produce a series of preparations using different extraction methods and to investigate quantitative and qualitative differences in their composition. Next, we studied anticancer activity of these preparations using selected in vitro and in vivo models. Several human and mouse cancer cell lines were used to investigate antiproliferative activity of the preparations and the selected ones were then studied for toxicity and antitumor effect in the in vivo mouse model of Lewis lung carcinoma.

Materials and methods

Plant material

The bark of U. tomentosa originated in Peru was kindly supplied by Vilcacora tomianki Centre (tomianki, Poland). The general characteristics of this material were described previously (Pilarski et al. 2006). The voucher material is deposited at the Laboratory of Phytochemistry. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland.

Obtaining B/[W.sub.37], B/[W.sub.b], B/50[E.sub.37], B/[E.sub.b] and B/96[E.sub.37] preparations

We prepared several extracts of 1.0 gram of the bark in 10 ml of water, 50%, or 96% ethanol for 8 h at 37 [degrees]C (B/[W.sub.37], B/50[E.sub.37] and B/96[E.sub.37], respectively). Water and 96% ethanol extractions were also performed at boiling temperatures of these solvents for 8 h (preparations B/[W.sub.b], and B/[E.sub.b]). Then, the extracts were centrifuged for 15 min at 4000 rpm. Supernatants were evaporated on Speed-Vac and next exsiccated with [P.sub.2][O.sub.5].

Obtaining alkaloids-rich bark preparation (B/SRT)

10.0 gram of the bark off. tomentosa was extracted with 50 ml of water (6 h, 37 [degrees]C). Next, the sample was centrifuged at 3500 rpm. 50 ml of dichloromethane was added to the supernatant and the mixture was intensively shaken. The organic layer was evaporated to dryness under vacuum at 40 [degrees]C. The residue (150 mg) was dissolved in 1.5 ml of 96% of ethanol and filtered through a 0.22 [micro]m filter.

HPLC-quantitative alkaloid analysis

The preparations were analyzed on oxindole alkaloid qualitative and quantitative contents according to the standardization protocols elaborated for U. tomentosa. To 100 mg of preparation, 15 ml of 2% sulphuric acid solution was added and sonified for 15 min with an ultrasonic bath (Bandelin Sonorex RK 103H). The mixture was then centrifuged at 3000 rpm for 10 min and extracted three times with 10 ml of ethylacetate. The aqueous phase was separated and adjusted to pH = 10 with 10% ammonium hydroxide and extracted three times with 10 ml of ethylacetate each. The organic phases were combined, evaporated to dryness and the residue dissolved in 1 ml of methanol. The qualitative and quantitative contents of alkaloids were determined by means of HPLC fingerprint analysis as desribed previously (Pilarski et al. 2007).

Cancer cell lines

Antiproliferative activity was evaluated using the following established in vitro cancer cell lines: HT-29 (colon adenocarcinoma), SW707 (human colorectal adenocarcinomas), KB (human cervical carcinoma), MCF7 (human breast carcinoma), A549 (human non-small cell lung carcinoma), OAW-42 (human ovarian cystoadenocarcinoma), HL60 (human acute promyelocytic leukemia), LLC (LL/2, mouse Lewis lung carcinoma) and B16 (mouse melanoma). All cell lines were obtained from the American Type Culture Collection (Rockville, Maryland, USA) and maintained at the Cell Culture Collection of the Institute of Immunology and Experimental Therapy, Wroclaw, Poland. 24 h before adding the test preparations, the cells were plated in 96-well plates (Sarstedt, USA) at a density of [10.sup.4] cells per well in 100 [micro]l of culture medium. The SW707, OAW-42, B16, LL/2 cell lines were cultured in the Dulbecco's Modified Eagle's Medium supplemented with 2 mM glutamine (DMEM, Gibco, Warsaw, Poland). The KB, MCF7, A549 cell lines were cultured in the Minimum Essential Medium Eagle (MEM, Gibco, Warsaw, Poland). The HL60 and HT-29 cell lines were cultured in the RPMI-1640 medium (Gibco, Warsaw, Poland) and McCoy's 5 medium supplemented with 2 mM glutamine (Gibco, Warsaw, Poland), respectively. All mediums were also supplemented with 10% fetal calf serum (Gibco, Grand Island, USA). The cell cultures were maintained at 37 [degrees]C humid atmosphere saturated with 5% [CO.sub.2].

Antiproliferative assays in vitro

The in vitro cytotoxic effect of B/[W.sub.37], B/[W.sub.b], B/50[E.sub.57], B/[E.sub.b], B/96[E.sub.37] and B/SRT was examined after 72-h of exposure of the cultured cells to varying concentrations of the preparations, using the SRB assay as described by Skehan et al. (1990). Briefly, the cells were attached to the bottom of plastic wells by fixing them with cold 50% trichloroacetic acid (TCA, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) on the top of the culture medium in each well. The plates were incubated at 4 [degrees]C for 1 h and then washed five times with running water. The background optical density was measured in the wells filled with culture medium without the cells. The cellular material fixed with TCA was stained with 0.4% sulforhodamine B (Sigma-Aldrich Chemie Gmbh, Steinheim, Germany) and dissolved in 1% acetic acid (POCH, Gliwice, Poland) for 30 min. Unbound dye was removed by rinsing (4x) with 1 % acetic acid. The protein-bound dye was extracted with 10mM unbuffered TRIS base (POCH, Gliwice, Poland) for determination of optical density (at 540 nm) in a computer-interfaced, 96-well microtiter plate reader Multiskan RC photometer (Labsystems, Helsinki, Finland). The in vitro results were presented as inhibitory concentration 50% ([IC.sub.50]) values. Each compound at a given concentration was tested in triplicates in each experiment, which was repeated two times.

Animal experiments

[C57Bl/6 x DBA/2][F.sub.1]([BDF.sub.1])male, 12-16 weeks old mice, weighing 20-25 g, supplied by the Department of Genetics and Laboratory Animal Breeding, M. Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland, were maintained in standard laboratory conditions. All experiments were performed according to Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing and Education issued by the New York Academy of Sciences Ad Hoc Committee on Animal Research and were approved by the 1st Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland.

Therapeutic efficacy of the B/[W.sub.37] preparation in different administration schedules to mice bearing LLC tumors

The B/W37 preparation was dissolved in physiological salt solution with the addition of 10% DMSO to final concentration of 25 mg/ml. Lower doses were prepared using serial dilutions. LLC cells derived from in vitro culture were inoculated subcutaneously (s.c.) into the right flank region with 3 x 105 cells per mouse (day 0). On 'days 1-21' animals were treated intraperitoneally (i.p.) with B/W37 preparation at doses of 5 mg/day (n = 5, one mouse died due to the trauma during the experiment), 0.5 mg/day (n = 6), 0.05 mg/day (n = 6) in the 200 [micro]l volume. One group (n = 6) received therapy by the preparation at the dose of 0.05 mg/day from 'days -7 to 21'. Two groups were treated with equivalent volume of physiological salt solution (control, n = 6) and DMSO 10% (n = 6). Animals were monitored for food and water consumption, weight, and general behavioral status.

Therapeutic responses of mice bearing LLC tumors on the B/96[E.sub.37] and B/SRT intraperitoneal injections

5 mg amounts of B/96E37 and B/SRT were dissolved in 200 [micro]l DMSO and filled up to 2 ml with the physiological salt solution. LLC cells derived from in vitro culture were inoculated subcutaneously (s.c.) into the right flank region with 2 x 105 cells per mouse (day 0). At the days 2-21 animals were treated i.p. B/96[E.sub.37] (n = 8) and B/SRT (n = 8) with a doses 0.05 mg/day and 0.5 mg/day (the control mice (n = 8) were injected with 200 [micro]l of the physiological salt). Animals were monitored for food and water consumption, weight, and general behavioral status.

Tumor cell cycle analysis

LLC lung carcinoma cells from primary tumors of mice treated by on the B/96[E.sub.37] and B/SRT were obtained after finishing the experiment by mincing fresh tumor tissue with a scalpel, passing them through a plasma filter, and suspending in phosphate-buffered saline (PBS). The cell suspension was washed once with PBS supplemented with 2% of fetal bovine serum. Then the cells were washed in PBS and counted in a hemacytometer. 1 x [10.sup.6] cells were fixed for 24 h in 70% ethanol at -20 [degrees]C. Next, the cells were washed twice in PBS and incubated with RNAse (50 [micro]g/ml, Fermentas, Germany) at 37 [degrees]C for 1 h. The cells were stained for 30 min with propidium iodide (50 [micro]g/ml, Sigma-Aldrich Chemie GmbH, Steinheim, Germany) at 4 [degrees]C and cellular DNA content of the samples was measured using the FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA). The results were analyzed using WinMDI 2.8 software.

Data processing and statistical analysis

The tumor volume was calculated using the formula TV = (a2 x b)/2, where 'a' means shorter diameter and 'b' means longer diameter in millimeters. Tumors were collected when the animals were sacrificed 22 days after the inoculation of the LLC cells. Dynamics of tumor growth in animal experiments were analyzed using the multiple analysis of variance (MANOVA) for the B/W37 preparation and one-way analysis of variance (ANOVA) for B/96E37 and B/SRT preparations. Blood morphology and cell cycle were assessed using ANOVA. Data transformations were applied to meet assumptions of parametric tests whenever applicable. Values of p < 0.05 were considered to be significant.

Results and discussion

HPLC-fingerprint analysis of alkaloids

The oxindole alkaloids profiles of all preparations used in cytotoxicity evaluations were presented in Table 1.
Table 1
The oxindole-alkaloid profiles of the studied preparations determined
by HPLC [expressed as percentages and in mg/100g of the preparations].

             Alkaloid                         B/[W.sub.37]

                                           mg            %

Uncarine F                           28 [+ or -] 1.4    6.59
Speciophylline                       68 [+ or -] 2.3   15.89
Mitraphylline                        33 [+ or -] 4.2    7.74
Pteropodine and isomitra-phylline  230 [+ or -] 12.5   53.44
Isopteropodine                       70 [+ or -] 0.9   16.34
Total                                      430        100

              Alkaloid                        B/[W.sub.b]

                                          mg            %

Uncarine F                          39 [+ or -] 3.2    7.89
Speciophylline                      32 [+ or -] 2.1    6.60
Mitraphylline                       13 [+ or -] 0.2    2.58
Pteropodine and isomitra-phylline  249 [+ or -] 7.9   50.88
Isopteropodine                     157 [+ or -] 5.6   32.05
Total                                     509        100

                Alkaloid                     B/5O[E.sub.37]

                                          mg             %

Uncarine F                         131 [+ or -] 5.3     7.12
Speciophylline                     217 [+ or -] 8.2    11.79
Mitraphylline                      151 [+ or -] 1.9     8.22
Pteropodine and isomitra-phylline  987 [+ or -] 11.2   53.62
Isopteropodine                     354 [+ or -] 23.2   19.25
Total                                         1896.8  100

                Alkaloid                         B/[E.sub.b]

                                            mg            %

Uncarine F                           303 [+ or -] 2.4    9.21
Speciophylline                        78 [+ or -] 5.6    2.38
Mitraphylline                        223 [+ or -] 8.3    6.79
Pteropodine and isomitra-phylline  1855 [+ or -] 89.0   56.38
Isopteropodine                      830 [+ or -] 14.4   25.24
Total                                          3408.7  100

               Alkaloid                        B/96[E.sub.37]

                                            mg             %

Uncarine F                           322 [+ or -] 10.3    9.26
Speciophylline                       553 [+ or -] 9.12   15.89
Mitraphylline                       322 [+ or -] 23.2     9.24
Pteropodine and isomitra-phylline  1782 [+ or -] 14.3    51.21
Isopteropodine                      501 [+ or -] 37.2    14.40
Total                                          3581.12  100

             Alkaloid                                 B/SRT

                                            mg              %

Uncarine F                          2712 [+ or -] 123.3    5.38
Speciophylline                     10937 [+ or -] 342.2   21.7
Mitraphylline                       8014 [+ or -] 271.2   15.9
Pteropodine and isomitra-phylline   19303 [+ or -] 54.9   38.3
Isopteropodine                      9435 [+ or -] 541.0   18.72
Total                                           51733.6  100


All of these preparations (B/[W.sub.37], B/[W.sub.b], B/50[E.sub.37], B/[E.sub.b], B/96[E.sub.37], and B/SRT) were produced from the same bark material. However, it might be easily expected that using different concentration of ethanol or applying dichloromethane in case of B/SRT would result in their chemical composition being significantly different. In fact, the alkaloid participation increases with the ethanol concentrations reaching 3.48% for the B/96E37 preparation obtained by extraction with nearly pure ethanol. The highest alkaloid content was shown for B/SRT where over 50% of dry mass was pure oxindole alkaloids. On the other hand, extraction with water results in the lowest alkaloid content of 0.43% observed for B/W37. The temperature of extraction was generally not significant for total alkaloid contents, however, this parameter was pivotal to quantitative representations of particular alkaloids. In general, the preparations obtained by extraction in boiling solvents have a lower speciophylline content and a higher contribution of isopteropodine. For instance, taking into account speciophylline, using boiling ethanol caused a decrease in its participation in total alkaloid content by over 6 times in comparison with the preparation obtained at 37 [degrees]C This observation is in agreement with literature in which non-stability of speciophylline is reported (Keplinger et al. 1999; Laus 1998; Laus et al. 1996). The above-presented changes in alkaloid profiles are probably a result of isomerisation by retro-Mannich ring opening, rotation and Mannich ring closure (Keplinger et al. 1999).

It must be emphasized that all presented fingerprints did not show the presence of any tetracyclic oxindole alkaloids confirming the high pharmacological quality of the obtained preparations (Lukasiak et al. 2001). A number of studies have shown that U. tomentosa occurs in nature in two different chemotypes characterized by pentacyclic (POA) or tetracyclic (TOA) pattern of indole and oxindole alkaloids (Hemingway and Phillipson 1974; Keplinger et al. 1999; Laus and Keplinger 1997). As shown, POA are more biologically active than TOA, particularly if their anti-inflammatory and antiproliferative potency is considered (Wurm et al. 1998). Both groups of alkaloids may act antagonistically and the selection of proper cat's claw material seems to be pivotal for a successful therapeutic approach (Keplinger et al. 1999).

Comparison of our HPLC determinations with chemical analyses of other cat's claw preparations available on the phytopharmaceutical market is not possible due to the lack of literature data. According to the standards informally adopted in Western Europe the concentration of oxindole alkaloids determined in relation to pteropodine should be below 1.75% in medicinal preparations (Lukasiak et al. 2001). This criterion is met only by the B/[W.sub.37], B/25[E.sub.37] preparations and possibly B/5OE37. Preparations with a higher content of this alkaloid such as B/[E.sub.b] and B/96[E.sub.37], and especially B/SRT - containing over 50% of alkaloids, should not be used according to these recommendations, due to the risk of overdose or side effects. On the other hand, this recommendation is not necessarily well-grounded, as the proposed method of standardization is not fully justified by the existing toxicological data which indicate that even pure oxindole alkaloids are safe to use. The LD50 value determined in mice which is higher than 16g per kg body mass (Keplinger et al. 1999) suggests that lethal poisoning of an adult person weighing 70 kg is hardly possible as it could occur after consuming 2 kg of the B/SRT preparation. It must be taken into account, however, that these animal data cannot be simply transferred to human beings.

Antiproliferative activity of preparations on various cancer cell lines

The obtained [IC.sub.50] values for all studied cell lines and preparations are presented in Table 2.
Table 2
[IC.sub.50] values for B/[W.sub.37], B/[W.sub.b], B/50[E.sub.37],
B/[E.sub.b], B/96[E.sub.37] and B/SRT [[micro]g/ml].

         Cell line                  [IC.sub.50] [[micro]-g/ml]

                              B/[W.sub.37]  B/[W.sub.b]  B/50[E.sub.37]

HT-29 (DSMZACC 136)               803.47       552.24        597.64
KB (ATCCCCL 17)                   417.19        81.43         59.22
LL/2 (LLC1) (ECACC 90020104)      461.26       234.15         75.72
B16 (4A5) (ECACC 94042254)        732.41       479.27        427.08
MCF 7                           >1000          759.19        631.60
A-549 (ATCCCCL 185)               566.71       550.42        404.06
OAW-42 (ECACC 85073102)           734.60       670.00        602.02
SW707                             419.10       378.22        320.00

           Cell line                 [IC.sub.50] [[micro]-g/ml]

                              B/[E.sub.b]  B/96[E.sub.37]   B/SRT

HT-29 (DSMZACC 136)             514.78         499.05      423.13
KB (ATCCCCL 17)                  41.64          35.69       23.57
LL/2 (LLC1) (ECACC 90020104)     39.16          25.06      246.79
B16 (4A5) (ECACC 94042254)      273.29         205.65      313.82
MCF 7                           416.32         375.60       29.86
A-549 (ATCCCCL 185)             168.65          93.17       40.03
OAW-42 (ECACC 85073102)         594.28         457.87      313.72
SW707                            67.41          49.06      133.67


Various cytotoxicities of the preparations related to their total alkaloid content were observed. The presented results demonstrated the highest antiproliferative activity for B/96[E.sub.37] and B/SRT whereas the lowest one for B/W37 (total alkaloid content of these preparations in 100g d.m. are 3480 mg, 50,401 mg and 430 mg, respectively). Our expectation that the highest - over 50% oxindole alkaloid content of this preparation - should correlate with the lowest IC50, was not fully confirmed. In fact, B/SRT was shown to have an inconsistent activity that can be explained by increased selectivity of this preparation for some cell lines or its low solubility in water and problems with reliable [IC.sub.50] determinations.

Differentiated activity of the preparations according is not surprising and was confirmed in our previous paper (Pilarski et al. 2007), whereas finding a correlation between IC50 values and cancer cell types is difficult and requires more studies. In case of B/96[E.sub.37] the highest growth inhibition was observed for Lewis lung carcinoma (LL/2) [[IC.sub.50] = 25.06 [micro]g/ml], cervical carcinoma (KB) [[IC.sub.50] = 35.69 [micro]g/ml] and colon adenocarcinoma (SW707) [[IC.sub.50] = 49.06 [micro]g/ml]. On the other hand, colon adenocarcinoma (HT-29), ovary cystoadenocarcinoma (OAW-42) and breast carcinoma (MCF-7) were the most resistant to this preparation [[IC.sub.50] = 499.05 [micro]g/ml, [IC.sub.50] = 457.87 [micro]g/ml, IC50 = 375.60 [micro]g/ml, respectively]. Other results were obtained for B/SRT that mainly inhibited proliferation of cervical carcinoma (KB) [[IC.sub.50] = 23.57 [micro]g/ml], breast carcinoma (MCF-7) [[IC.sub.50] = 29.86 [micro]g/ml] and lung carcinoma (A-549) [[IC.sub.50] =40.03 [micro]g/ml]. However, this preparation was less efficient than B/96E37 for Lewis lung carcinoma (LL/2) [IC50 = 246.79 [micro]g/ml] and colon adenocarcinoma (SW707) [[IC.sub.50] = 133.67 [micro]g/ml].

Comparison of the data obtained with other investigations is not easy due to the limited availability of IC50 determinations of U. tomentosa preparations and constituents. In one available study (Riva et al. 2001), [IC.sub.50] of U. tomentosa extracts fractionated on Sephadex and determined on breast carcinoma (MCF-7) ranged from 10 to 270 [micro]g/ml, and these values are similar to the results obtained in this study for B/SRT and B/96[E.sub.37] (29.86-375.60 [micro]g/ml, respectively). The authors of the cited paper do not indicate any constituents responsible for the observed cytotoxic properties except for the conclusion that they must be more soluble in methanol than in water, which may suggest oxindole alkaloids. Anyway, their results are in agreement with ours and suggest that ethanol preparations have increased activity as compared to aqueous ones (Riva et al. 2001).

We also compared our results with [IC.sub.50] obtained by other authors for pure oxindole alkaloids. These [IC.sub.50] values ranged from [10.sup.-5] to [10.sup.-4] mol for HL-60 and human leukemic monocytes (U-937) (Stuppner et al. 1993) and from 30 to 40 [micro]g/ml for human skin melanoma (SK-MEL), cervical carcinoma (KB), human breast carcinoma (BT-549), human ovarian adenocarcinoma (SK-OV-3) and monkey kidney epithelial (VERO) cells (Muhammad et al. 2001). Similar results were obtained for alkaloids isolated from U. guianensis that were tested on non-transformed mouse fibroblasts (BALB/c 3T3), human lung carcinoma (H460), cervical cancer (ME180), mice reticular lymphosarcoma (LSR), human prostate cancer (DU145) and mouse stomach carcinoma (C678). Importantly, in all these evaluations, isopteropodine was more active than pteropodine ([IC.sub.50] = 17-42 [micro]g/ml vs. [IC.sub.50] = 38-51 [micro]g/ml, respectively) (Lee et al. 1999). Another cytotoxicity assay was conducted on wild-type (RS188N) and engineered yeasts that lack the RAD52 DNA repair pathway, one of the three major DNA repair pathways that have been defined in yeast (RS321, RS322) [pteropodine: [IC.sub.12] = 140 [micro]g/ml (RS321), [IC.sub.12] = 680 [micro]g/ml (RS322), [IC.sub.12] [greater than or equal to] 8000 [micro]g/ml (RS188N); isopteropodine: [IC.sub.12] = 120 [micro]g/ml (RS321), [IC.sub.12] = 364 [micro]g/ml (RS322), [IC.sub.12] [greater than or equal to] 8000 [micro]g/ml (RS188N)] (Lee et al. 1999). The demonstrated selectivity of the alkaloids against the yeast mutants should be taken carefully due to the limitations of these evaluations in reference to [IC.sub.50] of mammalian cell lines.

Therapeutic effect of the B/[W.sub.37] preparation in mouse Lewis lung carcinoma model

The therapeutic activity of the B/[W.sub.37] preparation has been studied in mouse Lewis lung carcinoma model. Mice bearing LLC tumors were treated using different doses of the preparation during entire duration of the experiment. We found that mice which received B/[W.sub.37] preparation at the doses of 5 and 0.5 mg/day starting from 'day 1' of the experiment had statistically significant inhibition of tumor growth when compared to the rest of the groups (MANOVA, p = 0.0009, Fig. 1).

[FIGURE 1 OMITTED]

Morbidity and body weight of experimental animals during the run of the experiment were monitored. No animals died due to the therapy and we only observed a slight decrease in body weight for approximately 10% in the group treated with the highest 5 mg/day dose of the B/[W.sub.37] preparation (Fig. 2).

[FIGURE 2 OMITTED]

In the last day of the experiment blood samples were collected for morphology tests. The two groups treated with DMSO and B/[W.sub.37] at 0.05 mg/day, had significantly lower leukocytes numbers than the rest of the groups (ANOVA, p = 0.00002, Table 3). On the contrary, we have not found any significant differences in erythrocytes, platelets or hemoglobin between the experimental groups (data not shown).
Table 3
Leukocytes in the blood samples from mice bearing Lewis lung carcinoma
and treated with B/W37 extract at different doses and schedules.

                       Group                          Leukocytes
                                                 [[10.sup.3]/[micro]l]

Control                                          16.44 [+ or -] 9.36
DMSO                                              8.87 [+ or -] 1.56 **
B/[W.sub.37], 5 mg/day, started at 'day 1'       17.80 [+ or -] 2.80
B/[W.sub.37], 0.5 mg/day, started at 'day 1'     17.28 [+ or -] 11.26
B/[W.sub.37], 0.05 mg/day, started at 'day V      9.47 [+ or -] 2.14 **
* B/[W.sub.37], 0.5 mg/day, started at 'day -7'  14.37 [+ or -] 3.85

* The preparation name with asterisk (* B/[W.sub.37]) indicates start
of treatment on 'day -7'.
** These groups had significantly lower leukocytes numbers than the
rest of the animals (p = 0,00002).


The obtained results of blood parameters indicate that B/[W.sub.37] is well tolerated and non-toxic for the used doses. It is worth noting that the decrease in leukocyte count observed in the present study only partially confirms the results obtained in our previous experiments on chicken embryos, in which no significant differences in blood parameters were found, with the exception of MCV, MCH and MCHC (Pilarski et al. 2009). Furthermore, a number of data that are not in the accordance with the presented results can be found in the literature. The results of research on immunomodulatory properties of U. tomentosa that encompassed the analyses of selected blood parameters in calves after inducing local pneumonia can serve as an example here. In this experiment, a significant decrease in the number of thrombocytes was found in the group receiving cat's claw (Bednarek et al. 2002, 2004). Many investigations reported increased leukocyte numbers as well (Wagner et al. 1985; Wurm 1997; Wurm et al. 1998). For example the increase in leukocyte population was found in mice which were administered the C-Med-100 [R] preparation following the induction of leukopenia by means of doxorubicin (Sheng et al. 2000). Stimulation of immune system following the administration of U. tomentosa preparation was observed in a mouse model of listeriosis infection (Eberlin et al. 2005). Finally, an increase in the leukocyte content was found in the spleen in one study of effect of C-Med-100 [R] on healthy C57/BL/6 mice (Akesson et al. 2003b). B/96[E.sub.37] and B/SRT preparations lack antitumor activity in mouse Lewis lung carcinoma model

We also checked the activity of B/96[E.sub.37] and B/SRT preparations in a mouse Lewis lung carcinoma model. Both preparations were tested at the doses of 0.05 and 0.5 mg/day. As opposed to the B/[W.sub.37] preparation, B/96[E.sub.37] and B/SRT did not reveal any significant antitumor activity. No animals died due to the therapy and we did not observe a decrease in body weight of the animals treated with these preparations (data not shown).

Tumor cell cycle analysis

The results of the cell cycle analysis in tumor cells are unclear and need to be verified by a larger study. There were no significant changes in the cell cycle distributions with the exception of cell decrease at the [G.sub.2]/M phase after the administration of B/96[E.sub.37] at a daily-dose of 0.5 mg and the [G.sub.1]/[G.sub.0] cells cycle arrest demonstrated after the B/SRT therapy at a daily-dose of 0.05 mg (Fig. 3). The inhibition of the cells extracted from the tumor at these phases can constitute a response to stressogenic factors and be a result of the extended time of DNA repair (phase [G.sub1].) or a considerable inhibition of anabolic processes and cell adaptation to the unfavorable environmental conditions (phase [G.sub.0]). Such a cell-cycle arrest is very often a response of tumor cells on cytostatic drugs and one of the most frequent reasons of failure in chemotherapies because cancer cells may continue their proliferative activity after withdrawal of the drug (most chemotherapeutic agents induce apoptosis selectively in replicating cells). Therefore, the observed [G.sub.0]/[G.sub.1] cell accumulation may be a good explanation of lower effectiveness of B/SRT than expected from [IC.sub.50] determinations. This explanation remains unsatisfactory taking into consideration normal distribution of cell-cycles in case of tumors exposed to higher (0.5 mg/day) dose of B/SRT. Furthermore, it has been shown recently that oxindole alkaloids exerted cytotoxic effect on acute leukaemic lymphoblastic cells by induction of apoptosis in both, proliferating and [G.sub.0]/[G.sub.1] arrested stages (Bacher et al. 2006).

[FIGURE 3 OMITTED]

Conclusions

The results obtained for therapeutic effectiveness of B/[W.sub.37], B/96[E.sub.37] and B/SRT showed different anticancer properties of the tested preparations. Our studies showed that B/[W.sub.37] was the most active preparation under in vivo conditions as it inhibited the development of tumors at p = 0.0009.

Comparing the in vivo results with the in vitro [IC.sub.50] values for LLC cells presented in Table 2, some significant differences need to be indicated. First of all, in the case of in vitro tests B/[W.sub.37] ([IC.sub.50] = 461.26 [micro]g/ml) was the least active preparation. This preparation was almost two times less active than B/96[E.sub.37] (IC50 = 25.06 [micro]g/ml) and that is why the results of the in vivo tests are rather surprising. A possible explanation of the discrepancies shown can be related to the availability of the chemical compounds introduced into the body. The B/96[E.sub.37] preparation, containing non-polar compounds are less assimilable under physiological conditions than B/[W.sub.37] obtained by means of water extraction.

On the other hand the explanation that anticancer activity of the U. tomentosa preparations is only associated with their total alkaloid content may be erroneous. Low and ambiguous activity of B/SRT containing mainly pure alkaloids suggest that other phy-tochemicals are also responsible for cat's claw pharmacological potency. Finding these synergistic compounds by fractionation of U. tomentosa preparations is the most important task in near future.

Acknowledgements

We would like to thank Dr. Dmitry Nevozhay for his technical assistance, statistical evaluations and thorough review of this manuscript. We are grateful for the generous supply of Uncaria tomentosa from Vilcacora tomianki Centre (tomianki, Poland).

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Abbreviations: B/[W.sub.37], bark extracted in water at 37 [degrees]C; B/[W.sub.b], bark extracted in boiling water; B/50[E.sub.37], bark extracted in 50% ethanol at 37 [degrees]C; B/[E.sub.b], bark extracted in boiling 96% ethanol; B/96[E.sub.37], bark extracted in 96% ethanol at 37 [degrees]C; B/SRT, bark extracted in water and dichloromethane.

* Corresponding author. Tel.: +48 61 852 85 03; fax: +48 61 852 05 32.

E-mail address: krysgul@ibch.poznan.pl (K. Gulewicz).

0944-7113/$ - see front matter [C] 2010 Elsevier GmbH. All rights reserved.

Radoslaw Pilarski (a), Beata Filip (b), Joanna Wietrzyk (b), Mieczyslaw Kuras (c), Krzysztof Gulewicz (a), *

(a) Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14 str., 61-704 Poznan, Poland

(b) Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland

(c) Department of Ecotoxicology, Warsaw University, Warsaw, Poland

doi:10.1016/j.phymed.2010.04.013
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Author:Pilarski, Radoslaw; Filip, Beata; Wietrzyk, Joanna; Kuras, Mieczyslaw; Gulewicz, Krzysztof
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EXPO
Date:Dec 1, 2010
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