Screening of Anticancer and Immunomodulatory Activities of Panamanian Plants

Abstract

Fifty-one extracts, from six Panamanian plants species selected on the basis of their toxicities against Artemia salina, belonging to five families and five genera, were screened for their in vitro anticancer (TK-10, UACC-62, MCF-7) and immunomodulatory activities [inhibition of complement, classical (CP) and alternative (AP) complement pathway and lymphoproliferation]. The results showed that 9 (17.6%) of 51 extracts presented anticancer activity against cancer cell lines, while 28 (54.9%) of 51 extracts stimulated normal spleen cells proliferation, and 25 (49.0%) inhibited in vitro lymphoid tumor cell growth. Finally, 7 (19.4%) and 10 (27.8%) out of 36 extracts showed high and moderate anticomplementary activity on CP, respectively. Moreover, out of 36 extracts, 1 (2.8%) produced higher inhibition while 4 (11.1%) showed moderate inhibition in the alternate pathway. The dichloromethane, methanolic and aqueous extracts from the leaf (3.0, 3.2, 4.2) and branch (3.5, 5.7, 5.8) of Trichospermum galeottii and from the leaf (2.4, 2.5, 5.6) and stem (2.3, 2.3, 4.7) of Sauraria yasicae were the most active immunomodulatory plants. The dichloromethane extract from the root of Morinda panamensis (8.7, 4.4, 6.3) was active against the three cancer cell lines, while the dichloromethane extract from the bark of Trichospermum galeottii(TK-10 = 3.4,UACC-62 = 9.4) and the bark of Morinda panamensis (UACC-62 = 5.5, MCF-7 = 5.9) as well as the methanolic extract from the root of Morinda rojoc (UACC-62 = 9.2, MCF-7 = 9.4) exhibited selectivity against two cancer cell lines.

Keywords:

• Anticancer
• anticomplementary
• CYTED
• immunomodulatory
• medicinal plants
• Panamanian flora
• tumor cells

Introduction

Natural products have been the mainstay of chemotherapy of cancer for the past 30 years. Most of them are obtained from plants or microorganisms, as the plant-derived drugs, vinblastine, vincristine, irinotecan, topotecan, etoposide, paclitaxel and other natural antibiotics, dactinomycin, bleomycin and doxorubicin are now in clinical use (Roberts, 1997; Mann, 2002). From the investigation of coral reefs, rainforests, and deep subsurface thermal vents, novel bioactive compounds have been reported (Pezzuto, 1997; Cragg et al., 1997, 1999; Shu, 1998; Scott et al., 1998; Seymour, 1999; Lee, 1999; Skehan et al., 1990; Christian et al., 1997; Clark, 1996).

Latin binomials

Species	Family
Aegiphila anomalaPittier	Verbenaceae
Morinda panamensis Seem.	Rubiaceae
Morinda rojoc L.	Rubiaceae
Myroxylon balsamum (L.) Harms	Fabaceae
Saurauia yasicae Loes.	Actinidaceae
Trichospermum galeottii (Turcz.) Kosterm.	Tiliaceae

As a result of the National Cancer Institute's program to screen plants as a source of anticancer agents, one of the most widely used drugs for ovarian and breast cancer, paclitaxel, was discovered. According to Cragg et al. (1999) and Mann (2002), more than 50% of anticancer drugs in use today are derived from natural sources. The flora of Panama is one of the richest in the world (Correa et al., 1998), and its potential as a source of novel bioactive molecules has not been fully studied.

This paper reports the results of an international cooperative project X.3 “Evaluation of plant biodiversity of Iberoamerican countries as a source of immunomodulatory and chemotherapeutic agents”, within the framework of the Iberoamerican Program of Science and Technology for Development, CYTED. The effect on tumor cell lines growth as well as the immunomodulatory activities on splenocytes proliferation and on classical and alternative pathways of complement system were used to evaluate the effect exerted by the different extracts.

Fifty-one extracts from six species of Panamanian plants were subjected to these screening tests, with the aim of selecting the most promising plant species for further bio-guided isolation of active compounds.

Materials and Methods

Plant material

Plants were collected from five different areas of Panama, Parque Nacional de Campana, El Valle de Antón, Costa Arriba de Colón, and Llano Cartí and Sardinilla. Their taxonomic identity was established by Prof. Mireya Correa, Director of the Herbarium of the University of Panama (PMA), where voucher specimens are deposited. Table 1 summarizes plant names, voucher numbers, place and dates of plant collection. Plant material from different parts was air-dried in shade, pulverized in a Wiley mill and stored in hermetically sealed plastic bags until analysis.

Table 1 Results of anticancer activity of panamanian plants.

		DCM (GI50) (µg/ml)			MeOH (GI50) (µg/ml)			AQ (GI50) (µg/ml)
SPECIES/PLANT PART	FAMILY/VOUCHER/PLACE OF COLLECTION/DATE	TK-10	UACC-62	MCF-7	TK-10	UACC-62	MCF-7	TK-10	UACC-62	MCF-7
Aegiphila anomala	VERBENACEAE
branch	FLORPAN 3000	>100	50	42	47	32	<1	>100	56	26
bark	P.N., A.C.	67	18	21	>100	62	80	>100	>100	>100
leaf	Panamá, 12/03/1998	>100	>100	78	>100	54	55	>100	65	88
Morinda panamensis	RUBIACEAE
root	FLORPAN 2992	8.7	4.4	6.3	71	42	44	>100	84	42
bark	V. A., Cocké, 07/03/98	50	5.5	5.9	2.7	4.7	18	>100	67	>100
Morinda rojoc	RUBIACEAE
root	FLORPAN 2997	>100	35	23	79	9.2	9.4	>100	47	55
leaf	C. A., Colón, 08/03/98	7	37	>100	>100	14	>100	>100	>100	<1
Myroxylon balsamum	LEGUMINOSAE
bark	FLORPAN 2989	35	13	6.6	>100	44	61	>100	65	61
wood	El Valle de Antón,	88	31	32	>100	60	66	>100	94	>100
leaf	Coclé, 07/03/1998	45	20	19	>100	65	80	>100	92	61
Saurauia yasicae	DILLENIACEAE
leaf	FLORPAN 3001	72	61	>100	12	>100	27	>100	56	96
branch	P.N., A.C.	>100	34	26	>100	46	38	>100	>100	>100
stem	Panamá 12/03/1998	94	60	26	>100	36	10	>100	>100	>100
bark		64	36	30	89	15	13	18	27	>100
Trichospermum galeottii	TILIACEAE
leaf	FLORPAN 2998	>100	22	36	>100	48	22	>100	38	21
branch	C. A., Colón, 08/03/98	34	26	23	>100	38	26	>100	>100	>100
bark		3.4	9.4	77	40	32	16	>100	99	62

DCM: Dichloromethane, MeOH: Methanol, AQ: Aqueous, P.N., A.C.: Parque Nacional, Altos de Campana, C.A.: Costa Arriba

V.A.: El Valle de Antón, GI₅₀: 50% inhibition of growth; BOLD: extracts with activity

TK-10 (kidney), UACC-62 (melanoma), MCF-7 (breast)

Chemicals

Sigma Chemical Company (St. Louis, MO, USA) and Merck (Darmstadt, Germany) were the primary sources of chemicals used in this study. Electrophoretic reagents were from Bio-Rad (Richmond, CA), RPMI and FCS from Gibco (Grand Island, NY), and l-glutamine from Life Technologies (Paisley, UK). Other reagents were of analytical grade. Veronal saline buffer (VSB), containing 5 mM veronal (Sigma Chemical Co., USA) and 150 mM NaCl at pH 7.35, served as a stock solution for VSB²⁺, containing 0.5 mM Mg²⁺ and 0.15 mM Ca²⁺, and for EGTA-VSB, containing 5 mM Mg²⁺ and 8 mM ethylene glycol-bis-(2-aminoethyl)-tetraacetic acid (Sigma Chemical Co, USA).

Preparation of extracts

Plant material was extracted successively with chloroform or dichloromethane and methanol. Aqueous extracts were prepared from a fresh lot of plant material. The extracts were concentrated in a rotavapor at < 40°C, and aqueous extracts lyophilized.

Animals

Normal 2 to 4 month-old Balb/C mice of either sex were raised in the animal colony at the School of Pharmacy and Biochemistry of the University of Buenos Aires. Animals were housed in temperature-controlled rooms and maintained on Cargill pellets and Water ad libitum. Animal studies were conducted in accordance with the NIH Guidelines for the care and Use of Laboratory Animals.

Normal murine splenocytes

Mice were killed by cervical dislocation and spleens removed aseptically. Single cells were prepared by mincing spleen fragments and pressing them through stainless 200 mesh filter in RPMI-C medium (supplemented with 10% heat-inactivated foetal calf serum (FCS), 2 mM l-glutamine, 20 mM Hepes buffer, 100 µg/ml penicillin, 150 µg/ml streptomycin and 5 mM 2-mercaptoethanol. After washing three-times, cells were resuspended in RPMI-C and cultured at 1 × 10⁶ cells/ml. Cellular viability was determined by the Trypan blue exlcusion test. At least 200 cells were counted and the percentage of viable cells was recorded.

Tumor cells

Three human tumor cell lines, obtained from kidney, melanoma and breast (TK-10, UACC-62 and MCF-7), were provided by Dr. Gordon Cragg of the National Cancer Institute, USA. LB lymphoid leukemia arose spontaneously in a 6-month-old male Balb/C mouse as a lymphocytic T-cell leukemia and was maintained by serial passages in the peritoneal cavity of syngeneic host. LB cells were obtained by intraperitoneal (i.p.) puncture under sterile conditions, then resuspended and maintained in RPMI-C medium at 1 × 10⁶ cells/ml. Cellular viability was determined by the Trypan blue exclusion test. At least 200 cells were counted and the percentage of viable cells was recorded.

Alternate and classical complement pathways

Fresh normal rabbit erythrocytes (RaE) were obtained from New Zealand white rabbits (Harlan Iberica, Spain) and maintained in Alsever's solution. Fresh sheep erythrocytes were obtained from the School of Veterinary Sciences (Universidad Autónoma de Barcelona, Spain) maintained in Alsever's solution and sensitized with anti-sheep erythrocyte antibodies (ShEA, ICN Pharmaceuticals Inc., USA) according to Klerx et al. (1983). Human pooled serum (HPS) was prepared in our laboratory from the blood of five healthy volunteers (extractions were done in the Medical Services of the University of Barcelona) and stored at − 80° C until use.

Biological assays

Anticancer activity

Anticancer activity was evaluted using the NCI screening protocol (Monks et al., 1991). The relative cell growth or viability in treated and untreated cells was measured and 50% inhibition of growth (GI₅₀) was calculated (Monks et al., 1991 1997; Boyd et al., 1995).

Cell proliferation assay

The effect of each extract on the proliferation of normal splenocytes either alone or stimulated with Con A (4 µg/ml) or murine LB tumor cells was evaluated according with Fernández et al. (1998). Briefly, cells were cultured in RPMI-C, in the presence of concentration ranges of extracts (1–100 µg/ml). After 24 h at 37°C in a humidified atmosphere of 5% CO₂ in air, cultures were pulsed with one µCi of ³H-thymidine/well (Du Pont, NEN Products, Boston, MA, USA) and maintained for an additional 18h period prior to harvest. Incorporated [³H]TdR was measured in a liquid scintillation beta counter. Results were expressed as the mean cpm of [³H]TdR incorporated in triplicate cultures. Each experiment was repeated at least five-times.

Stimulation index (SI) was calculated as indicated:

Extract concentration required for 50% inhibition of DNA synthesis (IC₅₀) was calculated from plots of percentage cell growth versus drug doses.

Inhibition of complement hemolytic activity

Classical (CP) and alternative (AP) complement pathway activities were determined in HPS. The tests were performed in 96-well microtiter plates with RaE and ShEA as target cells for CP and AP, respectively (Klerx et al., 1983). Serial two-fold dilutions of samples were made in VSB²⁺ (CP assay) or EGTA-VSB (AP assay) in U-welled microtiter plates. Immediately thereafter, HPS in VSB²⁺ (CP) or HPS in EGTA-VB (AP) were added to each well. After preincubation at 37°C for 30 min, a suspension of sensitized sheep erythrocytes (4 × 10⁸) (CP) or a suspension of uncoated rabbit erythrocytes (1,15 × 10⁸) (AP) were added. The plates were incubated at 37°C for 60 min (CP) or 30 min (AP). Subsequently, the plates were centrifuged for 2 min at 1250 × g. To quantify hemolysis, 50 µl of the supernatant were mixed with 200 µl of water in flat-bottom microtiter plates and the absorption at 405 nm was measured with a ELISA BIO RAD Model 550.

Controls in this assay consisted of similarly treated supernatants of erythrocytes incubated in water (100% hemolysis), in buffer (0% hemolysis) or in buffer supplemented with HPS (0% inhibition). Data were collected as mean from four samples IC₅₀ values were calculated.

Results and Discussion

Anticancer activity

The results of the in vitro anticancer screening of the six plant species are summarized in Table 2. As can be seen, 9 (17.6%) of 51 extracts tested showed activity against at least one of the three human tumor cell lines (GI₅₀ < 10 µg/ml). The dichloromethane extract from the root of Morinda panamensis Seem (Rubiaceae) had marked cytoxicity (GI₅₀: 8.7, 4.4, 6.3) against the three cell lines. Extracts with marked activity against two cell lines (TK-10 and UACC-62) were the dichloromethane extract from the bark of Trichospermum galeotti (Turcz.) Kosterm (Tiliaceae) (GI₅₀: 3.4, 9.4) and the methanolic extract from the bark of Morinda panamensis (GI₅₀: 2.7, 4.7). Similar results against two cell lines (UACC-62 and MCF-7) were obtained with the methanolic extract of the root of Morinda rojoc L. (GI₅₀: 9.2, 9.4).

Table 2 Results of immunomodulatory activity of panamanian plants.

		COMPLEMENT (IC50) (µg/ml)						IMMUNOMODULATORY ACTIVITY
											IC50(µg/ml)
		CP			AP			(SI) NMS			NMS + ConA			LB cells
SPECIES/PLANT PART	FAMILY/VOUCHER/DATE/PLACE OF COLLECTION	DCM	MeOH	AQ	DCM	MeOH	AQ	DCM	MeOH	AQ	DCM	MeOH	AQ	DCM	MeOH	AQ
Aegiphila anomala	VERBENACEAE
branch	FLORPAN 3000 P.N., A. C.	×	*	×	×	*	×	NE	NS	2.1	100	28.2	63.1	79.4	3	NE
bark		20.4	*	×	−	*	×	NE	NE	2.7	NS	45	NE	>100	69.2	NE
leaf	Panamá, 12/03/1998	85.2	401	481.5	−	−	−	NS	1.82	1.82	NS	79.4	NE	41.7	NS	NE
Morinda panamensis	RUBIACEAE
root	FLORPAN 2992	41.2	13.2	×	−	−	×	NS	1.8	2.7	19.1	>100	NE	40	83.2	NE
bark	V. A., Coclé, 07/03/98	59.8	4.8	×	−	−	×	NE	NS	NS	83.2	69.2	NE	40	>100	NS
Morinda rojoc	RUBIACEAE
root	FLORPAN 2997	1.4	9.6	−	−	−	−	NS	NS	NE	NE	10.2	NS	52.5	15.8	50.1
leaf	C. A., Colón, 08/03/98	34.1	5	350	−	−	−	2.5	NE	2.5	NE	NE	NE	50	NE	52.5
Myroxylon balsamum	LEGUMINOSAE
bark	FLORPAN 2989	×	140	×	×	−	×	1.5	NS	2.1	41.7	79.4	NE	79.4	>100	>100
wood	V. A., Cocle, 07/03/98	×	71,4	×	×	−	×	NS	NS	NS	NS	85.1	NE	NS	NS	NS
leaf		83.6	4,3	×	−	−	×	1.8	3.5	NS	100	NE	NE	NS	NS	NE
Saurauia yasicae	DILLENIACEAE
leaf	FLORPAN 3001	150	34,6	650	−	1300	860.5	2.4	2.5	5.6	NE	NE	NE	85,1	NS	NS
branch	P.N., A.C.	78.7	10	×	−	430	×	2	NE	2	NE	NE	NE	NE	NS	NE
stem	Panamá, 12/03/1998	78.4	17,4	×	−	195.6	×	2.3	2.3	4.7	NE	NE	NE	NS	NS	NE
bark		171.4	7,2	14,7	−	6.7	210.7	NE	NE	3.6	NE	NE	NE	26,3	22,4	NE
Trichospermum galeottii	TILIACEAE
leaf	FLORPAN 2998	87.5	20,9	×	−	241.4	×	3	3.2	4.2	NE	67.6	NE	NS	38	58.9
branch	C. A., Colón, 08/03/98	115	13.7	×	−	114.3	×	3.5	5.7	5.8	NS	NE	NE	100	NS	69.2
bark		−	710	×	−	−	×	NE	NS	6	93.3	47.9	NS	NS	1.9	NS

DCM: Dichloromethane; MeOH: Methanol; AQ: Aqueous; *: Extract with hemolytic activity; –: Extract without activity at range of concentrations assayed × : Not tested, NS: No significant effect, NE: No effect, SI: Stimulation Index, CP: Classical Pathway, AP = Alternate Pathway, ConA: Concavallin A, NMS: Normal Mouse Splenocytes, LB: Lymphocytes B; IC₅₀: 50% inhibition of DNA synthesis, P.N., A.C.: Parque Nacional, Altose de Campana, C.A.: Costa Arriba

V.A.: El Valle de Antón; BOLD: extracts with activity

A literature review using NAPRALERT and Science Finder database showed no information on Morinda rojoc, Saurauia yasicae Loes and Trichospermum galeottii. Of these, Morinda rojocand Trichospermum galeottii exhibited marked cytotoxicity against at least one cell line.

As shown in Tables 1 and 2, the plants showing anticancer activity as well as anticomplement effect on the classical or alternative pathway were the methanolic extract of the root of Morinda rojoc and the methanolic extract of the bark of Morinda panamensis. Trichospermum galeottii not only exhibited anticancer activity and anticomplement activity but also a high stimulatory effect on normal murine spleen cell proliferation.

Hemolytic assay on human complement activity

The effect of different extracts on the classical (CP) and alternative (AP) human complementpathways is shown in Table 3. Out of 36 extracts, 7 (19.4%) and 10 (27.8%) showed high and moderate anticomplementary activity on CP, respectively. Morever, 1 extract (2.9%) produced higher inhibition, while 4 (9.1%) showed moderate inhibition in alternate pathway.

The dichloromethane extract of the root of Morinda rojoc was the most active inhibitor of CP (IC₅₀ = 1.4 µg/ml). Moreover, the extracts that showed a strong anticomplement effect on CP were the methanolic extract of the leaf of Myroxylon balsamum and Morinda rojoc, and the methanolic extract of the bark of Morinda panamensis (IC₅₀ < 5 µg/ml).

The highest inhibitory effect on the AP was obtained with the methanolic extract of the bark of Saurauia yasicae (IC₅₀ = 65.7 µg/ml), which was also very active on CP (IC₅₀ = 7.2 µg/ml). The methanolic extract of the branch and leaf of Thichospermun galeottii, the methanolic extract of the stem and the aqueous extract of the bark of Saurauia yasicae have moderate activity on AP.

Immunomodulatory effects

The six species were also evaluated for their immunomodulating activity. Either normal mouse splenocytes or leukemic LB cells were used to evaluate the immunomodulatory activity of the different extracts. The results showed that 28 extracts (54.9%) were able to stimulate normal splenocytes growth while 25 (49%) inhibited LB tumor cell growth, while 17 (33.3%) also inhibited Con A stimulated normal spleen cells. Different degrees of activities were found. Dichloromethane, methanolic and aqueous extracts from the leaf and branch and aqueous extracts from wood of Trichospermum galeotti presented higher stimulatory effect on normal murine spleen cell proliferation. Similar results were found when dichloromethane, methanolic and aqueous extracts from the leaf or stem of Saurauia yasicae were assayed. Moreover, when LB tumor cells were used, these extracts inhibited tumor cell growth. Therefore, they were selected to continue with the bioguided studies.

The result given in this paper are a preliminary evaluation of the most interesting plants species collected and provides additional biological data on Panamanian plants. Currently, biological activity-guided isolation of the active constituents from the most active/selective plant extracts is ongoing.

Acknowledgements

This research was supported by the Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (CYTED), Organization of American States (O.A.S.) Project SEDI/AICD/AE/106/01, Secretaría Nacional de Ciencia y Tecnología e Innovación (SENACYT) and the International Cooperative Biodiversity Group (ICBG). This work was supported in part with grants from University of Buenos Aires, Argentina. Thanks are due to Dr. Gordon Cragg (National Cancer Institute) for supplying cell lines and Carlos Guerra and Alex Espinosa (University of Panama) for collection of plant materials and Paula Cerdá Zolezzi for technical assistance (University of Buenos Aires).