Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas ISSN: 0717-7917 [email protected] Universidad de Santiago de Chile Chile

Fazio, Ana Laura; Ballén, Diana; Cesari, Italo M.; Abad, María Jesús; Arsenak, Miriam; Taylor, Peter An ethanolic extract of Uncaria tomentosa reduces inflammation and B16- BL6 melanoma growth in C57BL/6 mice Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, vol. 7, núm. 4, julio, 2008, pp. 217-224 Universidad de Santiago de Chile Santiago, Chile

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ArtículoOriginal | Original Article

An ethanolic extract of Uncaria tomentosa reduces inflammation and B16- BL6 melanoma growth in C57BL/6 mice.

[Un extracto etanólico de Uncaria tomentosa reduce la inflamación y el crecimiento del melanoma B16/BL6 en ratones C57BL/6] Ana Laura FAZIO1, Diana BALLÉN2, Italo M. CESARI2, María Jesús ABAD1, Miriam ARSENAK1, Peter TAYLOR1 1. Centro de Medicina Experimental , Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020-A, Venezuela. 2. Centro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas, Apartado 20632, Caracas 1020- A, Venezuela. Contact : Tel +58 212 504 1097; Fax: +58 212 504 1086. E-Mail: [email protected]

Recibido | Received 4/06/2008; Aceptado | Accepted 30/06/2008; Online 12/07/2008

Abstract Extracts of the bark of Uncaria tomentosa (Cat’s Claw – Uña de Gato) have been used traditionally for their anti-inflammatory and anticancer properties. We investigated the effect of a hydroethanolic extract (UT) of U. tomentosa on a) the viability of primary and tumor cells, b) the inflammatory response (tumor necrosis factor alpha [TNF-α], interleukin-6 [IL-6] and nitric oxide [NO]) both in vitro and in vivo, c) B16/BL6 melanoma cell growth and metastasis in the C57BL/6 mouse, and d) nuclear factor κB (NF-κB) activity in LPS-stimulated HeLa cells. UT did not show an important cytotoxic effect in vitro at the doses up to 300 μg/ml, but did inhibit tumor growth and metastasis in vivo. UT inhibited TNF-α, IL-6 and NO production in vitro. NF-κB activity was also inhibited. Our studies show that UT merits further study for its effects on processes common to inflammation and cancer. Keywords: Uncaria tomentosa, cancer, NF-κB, inflammation, B16/BL6 mouse Resumen Los extractos de la corteza de Uncaria tomentosa (Uña de Gato) han sido usados tradicionalmente por sus propiedades anti-inflamatorias y antitumoral. Investigamos el efecto de un extracto hidroetanólico (UT) de U. tomentosa sobre: a) la viabilidad de células normales y tumorales, b) la respuesta inflamatoria (factor de necrosis tumoral alfa (TNF-a), interleuquina-6 (IL-6) y óxido nítrico (ON) in vitro e in vivo, c) crecimiento y metástasis de células de melanoma B16/BL6 en ratones C57BL/6, y d) actividad del factor nuclear κB (NF-κB) en células HeLa estimuladas con LPS. UT no mostró un efecto citotóxico importante in vitro hasta 300 µg/ml, pero si inhibió el crecimiento de tumor primario y de metástasis in vivo. UT inhibió la producción de TNF-α, IL-6 y ON in vitro. La actividad de NF-κB también resultó inhibida. Nuestros estudios muestran que UT contiene compuestos que ameritan ser más estudiados por sus efectos sobre procesos comunes a inflamación y cáncer. Palabras clave: Uncaria tomentosa, cáncer, NF-κB, inflammación, B16/BL6 INTRODUCTION and Werb, 2002) so a dual action against both inflammation and cancer is not surprising. Studies Uncaria tomentosa is a species of vine from the have shown that anti-inflammatory drugs may be Rubiaceae family, widely distributed throughout effective in cancer therapy and/or prevention (Thun South and Central America, which has a long et al., 2002), and the possible mechanisms of action tradition of use as a folk medicine for the treatment of of anti-inflammatory phytochemicals have been a variety of conditions including inflammation, reviewed (Surh et al., 2001). U. tomentosa extracts cancer and gastrointestinal disorders (Reinhard, have been shown to inhibit NF-κB (Sandoval-Chacón 1999). Previous studies have shown anti- et al., 1998; Akesson et al., 2003), a transcription inflammatory activity both in vitro (Sandoval-Chacón factor which represents an important link between et al., 1998) and in mouse models of inflammation chronic inflammation and cancer (Li et al., 2005) and (Aquino et al., 1991; Caballero et al., 2005), and which has been suggested as a possible target for the growth inhibitory effects in both tumor (Sheng et al., therapy of both (Bremner and Heinrich, 2002). 1998) and normal cells (Akesson et al., 2003) in We have previously shown that intraperitoneal vitro. The role of chronic inflammation in tumor (i.p.) injection of an aqueous extract of U. tomentosa initiation and growth is well established (Coussens

Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth Fazio et al. inhibited the TNF-α and IL-6 response to Female C57BL/6 mice (7–9 weeks old) were lipopolysaccharide (LPS) challenge, and the growth obtained from the Animal Facility, IVIC and fed with of both primary tumors and metastasis in mice standard pellet diet and water ad libitum. All animal (Caballero et al., 2005). In the present study, we experiments were performed according to report the in vivo anti-inflammatory and antitumor internationally accepted guidelines for the treatment activities of a hydroethanolic extract, as well as an of animals in research. inhibitory effect on NF-κB. Cytotoxicity. MATERIALS AND METHODS Cells were plated at 2.5 - 5 x 104 cells / well in 100 µl of culture medium in flat-bottomed 96 well Plant material. plates and allowed to attach for 24 h. Different The bark of Uncaria tomentosa (Cat’s claw, Uña concentrations of UT in 100 µl culture medium were de gato), was obtained from the Peruvian Amazon then added. Control wells were set up containing region and identified by us. Aliquots were ground equivalent quantities of ethanol, which in no case then macerated in a 70% ethanol in water solution for exceeded 1%. No effect was observed due to the 21 days in the dark at room temperature. The ethanol. After a further 24 h, the number of viable suspension was then filtered under sterile conditions cells was assessed using the MTS/PMS chromogenic using Whatman No. 1 filter paper then adjusted to a assay (Promega Corp., USA) according to the stock concentration of 5 mg/ml, which was calculated manufacturer’s instructions. from the dry weight of a lyophilized sample. This extract shall be termed UT. Inflammatory response in vitro. Peritoneal macrophages were activated with 10 Cells and animals. µg/ml lipopolysaccharide (LPS - E. coli serotype The cell lines, B16/BL6 (murine melanoma), 055:B5, Sigma, USA) for 24 h in the presence of UT, K1735 (amelanotic murine melanoma), HT29 and then the concentrations of TNF- α, IL-6 and (human colon carcinoma), A549 (human lung nitric oxide (NO) were measured in the supernatants. carcinoma), WEHI 164 (mouse fibrosarcoma), LEC TNF- α was quantified using the WEHI 164 cell (mouse liver endothelial cell line) were cultured in bioassay (Espevik and Nissen-Meyer, 1986), IL-6 Dulbecco’s Modified Eagle’s Medium (DMEM) with a commercial ELISA assay (R & D Systems supplemented with 10% heat-inactivated fetal bovine Inc., MN, USA) and NO using the Griess reaction serum (FBS - Gibco, BRL, USA), penicillin (100 (Sandoval-Chacón et al., 1998). Units/ml), streptomycin (100 μg/ml) and containing in addition glucose 0.45% (HT29 cells), and L- Inflammatory response in vivo. glutamine 2 mM (A549 cells). Human peripheral Mice were injected intraperitoneally (i.p.) with blood monocytes (huPBMC) were obtained from different doses of LPS in 100 μl of PBS. After 1 h, healthy donors by standard Ficoll/Hypaque gradient blood was collected by heart puncture under ether centrifugation and cultured in RPMI-1640 10% FBS. anesthesia. Serum was separated and assayed for the Chopped spleens from C57BL/6 mice were ground two cytokines and NO as described above. In order to through a wire mesh screen. After removal of detritus evaluate the effect of UT on the inflammatory and lysis of red blood cells with 0.085% sodium response, mice were injected i.p. with 50 μg UT on 3 citrate, adherent cells were removed by overnight consecutive days prior to LPS challenge. incubation in plastic culture flasks. The non-adherent cells (muSplen) were harvested, counted and cultured Lung metastasis. in RPMI-1640 10% FBS. Murine peritoneal At day 0, mice were inoculated in the lateral tail macrophages (muPM) were collected from C57BL/6 vein (i.v.) with 105 B16/BL6 cells in 100 µl PBS. mice 4 days after a peritoneal injection of 2 ml of 4% Two treatment protocols with UT were performed a) thioglycollate. The cells were washed, seeded into intraperitoneal (i.p.) injection of 50 μg of UT in 100 culture flasks in RPMI-1640 10% FBS, and non- µl PBS / 25% ethanol on days -2, -1 and 0, and b) adherent cells discarded after 3 h. The adherent cells i.p. injection of the same dose of extract 5 times per were then used immediately. week starting from on day 0 up to day 21. Control

Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008 218 Uncaria tomentosa reduces inflammation and B16-BL6 melanoma growth Fazio et al. animals received 100 μl PBS / 25% ethanol. On day the higher concentrations of 100 and 300 μg/ml. 23, the animals were sacrificed with ether; the lungs Inhibition was less than 50% in all cases. Three were removed, placed for 5 min in 3% H2O2 in H2O tumor cell lines (HT-29, K1735 and WEHI) and the and fixed in Bouin's solution. The purpose of the three primary cell preparations (huPBMC, muSplen H2O2 was twofold: to bleach hemorrhages which and muPM) were more sensitive to the extract than could be mistaken for metastases, and to inflate the the B16/BL6, A549 and LEC cells. lungs, facilitating the evaluation of metastases under the dissecting microscope. Animals were challenged with LPS prior to sacrifice, in order to measure serum TNF-α and IL-6 levels as described above.

Primary tumors. Primary tumors were induced by the subcutaneous (s.c.) injection of 5 x 104 B16/BL6 cells in 100 µl PBS into the hind limb. The mice were injected i.p. with 50 μg of UT 5 times per week starting from on day 0 up to day 21. Tumor size was measured in two dimensions with a vernier gauge. Animals were challenged with LPS prior to sacrifice, in order to measure serum TNF-α and IL-6 levels as described above. Figure 1. Effect of the plant extracts on the viability of tumor cell lines in vitro. Cell viability was measured by the MTS chromogenic assay after a 24 h incubation in the presence of UT NF-κB luciferase assay. at the concentrations indicated. huPBMC - human peripheral HeLa cells were transiently transfected with the blood monocytes, muSplen – non-adherent mouse splenocytes, NF-κB luciferase reporter system (Stratagene, La muPM - murine peritoneal macrophages. Results are expressed as the percentage of the absorbance in the absence of the plant Jolla, CA, USA) according the manufacturer’s extract (mean ± S.E.M., n = 3). instructions. Transfected cells were seeded into 96- well plates, allowed to adhere overnight then treated Effect of UT on the inflammatory response in for 1 h with different concentrations of UT. The cells vitro and in vivo. were then stimulated for a further 4 h with 25 ng/ml The TNF-α response of mouse peritoneal huTNF-α (BD Biosciences, Palo Alto, CA, USA). macrophages to LPS was reduced by 48% in the Luciferase activity was measured using the Steady- presence of 100 μg/ml UT (Fig. 2), although this Glo assay kit (Promega, Madison, WI, USA), in a 96- reduction was not significant due to variability in the well luminometer. Dexamethasone and paclitaxel, results. The IL-6 and NO responses were which are known to inhibit and stimulate NF-κB significantly reduced by 35% and 62% respectively activity respectively, were included as controls. (P < 0.001). This result was not due to a direct Statistical analysis. cytotoxic effect, as no change in the viability of these activated cells was observed at this concentration of Each experiment was performed at least three UT (results not shown). A similar reduction in the times and results are expressed as the mean ± inflammatory response was observed in vivo, when standard deviation. The unpaired Student’s t test with mice were injected i.p. with 50 μg UT on 3 the Welch correction was used to assess the statistical consecutive days prior to challenge with different significance of the differences. A confidence level of doses of LPS (Fig. 3). The TNF, IL-6 and NO P < 0.05 was considered significant. responses were reduced by 63, 59 and 57% respectively when the animals treated with UT were RESULTS challenged with the highest dose of LPS. Again, only the results for IL-6 and NO were significant due to Cytotoxicity. variability in the TNF response to LPS. Initial in vitro experiments (Fig. 1) showed an inhibitory effect of UT on 24 h cell growth only at

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Figure 3. Inhibition by UT of the inflammatory response to LPS in mice. Animals were pretreated with 50 μg/ml UT i.p. on 3 days, then challenged with different doses of LPS. After 1 h, blood was extracted and the serum assayed for TNF- α, IL-6 and Figure 2. Inhibition by UT of the inflammatory response of NO. (mean ± S.E.M., n = 3). * P < 0.05. mouse peritoneal macrophages to LPS. Cells were activated with 10 µg/ml LPS for 24 h in the presence of 100 μg/ml UT extract. Figure 5 shows that both the TNF-α and IL-6 TNF- α, IL-6 and NO levels were then measured in the responses to low dose LPS were much greater in supernatants. (mean ± S.E.M., n = 3). ** P < 0.001 animals with primary tumors than in animals without tumors (compare with Fig. 3). In contrast, very little Inhibition by UT of primary tumor growth and priming of the inflammatory response to low dose metastasis in mice. LPS was observed in the animals with metastasis. Mice were inoculated s.c. with B16/BL6 cells and The TNF-α and IL-6 responses in animals with the effect of i.p. UT on primary tumor growth was primary tumors were greatly inhibited by UT measured. At all time points after the appearance of treatment, (85% and 81% respectively). Although the tumor, there was a very significant inhibition of TNF-α levels were lower in the untreated animals tumor growth in the animals treated with UT (Fig. with metastasis, an important inhibitory effect (81%) 4A). This effect was most notable at earlier times was observed after UT treatment. The already almost with the treated tumors measuring approximately basal levels of IL-6 in these animals were unaffected 75% less than the controls up to day 16, but only 50% by UT. less at day 22. Effect of UT on the inflammatory response to LPS in tumor-bearing animals. As basal levels of serum TNF-α and IL-6 are very low in animals with either primary tumors or metastases, we evaluated the effect of UT on the inflammatory response to a low dose of LPS (3 μg / animal) in tumor-bearing animals, prior to sacrifice.

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Figure 5. Effect of UT on the inflammatory response to LPS in tumor-bearing mice. C57Bl/6 mice were inoculated s.c. or i.v. with B16/BL6 tumor cells to produce primary tumors or metastases, respectively, then injected i.p. 5 times a week up to day 21 with 50 μg UT (corresponding to Fig 4A and 4C). One h before sacrifice, the animals were challenged with 3 μg LPS. Blood was extracted and the serum assayed for TNF- α and IL-6. (mean ± S.E.M., n = 3). * P < 0.05.

Effect of UT on the NF-κB response to activation by TNF-α in HeLa cells. The effect of UT on the NF-κB response of HeLa cells to TNF-α was determined in a luciferase reporter assay. UT at 10 μg/ml slightly stimulated Figure 4. Effect of UT treatment on primary tumor growth and NF-κB activity, but was inhibitory (47%) at 100 metastasis in mice. A. C57Bl/6 mice were inoculated s.c. with B16/BL6 tumor cells to initiate a primary tumor and injected i.p. μg/ml (Fig. 6). The highest dose of dexamethasone 5 times a week up to day 21 with 50 μg UT. Tumor growth was used (30 μM), a known NF-κB inhibitor, inhibited the assessed with a vernier gauge. B. Mice were inoculated i.v. with response by 38%. A change of medium after a B16/BL6 cells. Treatment with UT consisted of 50 μg i.p. and the preincubation with UT only slightly reduced the 3 days prior to inoculation. Lung metastases were counted on day degree of inhibition, suggesting that the effect was 22. C. Mice were inoculated i.v. with tumor cells, as in B., but treated with UT post-inoculation as in A. (mean ± S.E.M., n = not due to a direct interaction between the crude 10). * P < 0.005, ** P < 0.0005. extract and LPS or TNF-α (results not shown). Preliminary experiments also confirmed that UT was not cytotoxic for these cells at the doses used in these short term assays (results not shown).

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that an aqueous extract of this plant inhibited TNF-α and nitrite production by the RAW 264.7 mouse macrophage cell line in vitro (Sandoval et al., 2000; Sandoval et al., 2002), as far as we are aware this is the first report of its inhibitory effect on inflammatory cytokine production in vivo while there have been two reports of a stimulatory effect of Uncaria extracts on IL-6 production (Lemaire et al., 1999; Eberlin et al., 2005). Evidently such results must be interpreted taking into account the nature of the preparation used, and its route of administration. Aguilar reported that a hydroethanolic extract was more active than a water extract in the mouse paw edema assay (Aguilar et al., 2002). Such extracts may

Figure 6. Effect of UT treatment on NF-κB activity. HeLa cells be expected to contain compounds with a wider range transfected with the NF-κB/luciferase reporter plasmid were of polarities, including bioactive alkaloids, than a treated for 1 h with different concentrations of UT, then water extract but also less desirable compounds such stimulated for a further 4 h with 25 ng/ml huTNF-α. DEX – as tannins (Sheng et al., 2005; Pilarski et al., 2006). dexamethasone (30 μM), PAC – paclitaxel (1 μM) – see Materials and Methods.. (mean ± S.E.M., n = 3). Our finding that UT inhibits the inflammatory response as well as tumor growth and metastasis, in DISCUSSION the same mouse model is interesting considering the proven relationship between inflammation and Our previous studies indicated that an aqueous cancer. UT may contain compounds which separately extract of UT was not cytotoxic for a range of cells at exert anti-inflammatory or anticancer effects through concentrations up to 3 mg/ml (Caballero et al., 2005). different mechanisms, or the results may be due to a Here we show that the hydroethanolic extract of UT, compound, or group of compounds, acting through a was more cytotoxic for some of the tumor cell lines common mechanism. Plant components with both and the primary cells, but not for the B16/BL6 activities including flavonoids and terpenes have melanoma cells. We were unable to test been reported (Evans and Taylor, 1983; Middleton et concentrations higher than 300 μg/ml, as a precipitate al., 2000). In a previous study, we showed that began to appear in the cultures at 1 mg/ml. A review blocking TNF-α with a TNF receptor construct of several reports suggests that extracts of U. decreased serum TNF-α and IL-6 levels after LPS tomentosa may show some antiproliferative activity challenge in tumor-inoculated mice (Cubillos et al., (Sandoval-Chacón et al., 1998; Sheng et al., 1998; 1997) as well as reducing the number of lung Riva et al., 2001; Akesson et al., 2003; De Martino et metastases in the same animals. al., 2006) although the effects were observed in the Previous studies have shown that aqueous extracts “mg/ml” range. Santa María showed that aqueous of U. tomentosa inhibit NF-κB activity (Sandoval- extracts of U. tomentosa were not toxic for Chinese Chacón et al., 1998; Akesson et al., 2003), whereas hamster ovary cells (CHO) in 3 different bioassay Aguilar reported greater inhibitory activity in a systems (Santa Maria et al., 1997). In contrast, U. hydroethanolic extract (Aguilar et al., 2002). Here, tomentosa was reported to attenuate peroxynitrite- we confirm that our hydroethanolic extract was also induced apoptosis in HT29 and RAW 264.7 cells inhibitory for NF-κB, at concentrations that were not (Sandoval-Chacón et al., 1998). Considering these cytotoxic. However, NF-κB is an anti-apoptotic results and the dose of UT we used in the in vivo factor in cells and its constitutive expression may be experiments (50 μg UT/day), it is difficult to important in the survival of tumor cells (Aggarwal, conclude that the inhibitory effect seen with the 2004). Thus, the anticancer effects of anti- NF-κB primary tumors and metastasis was due to a direct compounds derived from plants may derive from an effect on tumor cell proliferation or viability. inhibition of inflammatory cells, as well as a direct Our results confirm previous reports of the anti- effect, at higher concentrations, on the tumor cells inflammatory action of extracts of Cat’s Claw themselves. (Aquino et al., 1991). Although it has been reported

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