Abstract
Venezuela is a country where indigenous and local communities use more than 1500 species of plants for medicinal purposes. Anticancer drugs derived from plants may act through a direct cytotoxic effect on tumor cells or through other mechanisms such as inhibitory effects on the proteases involved in tumor growth and spread. From our databases of botanical collections and ethnobiological usage, we selected 17 species to conduct an initial survey of cytotoxicity on eight human tumor cell lines (representing lung, breast, colon, and pancreas) and activity against four proteases. Thirteen extracts from 10 species were cytotoxic, at 100 μ.g/ml or less, on one or more cell lines. Extracts of Jacaranda copaia and Tapirira guianensis were active against several cell lines, whereas extracts of Gnetum nodiflorum, Protium heptaphyllum, Protium unifoliolatum, Costus scaber, and Croton cuneatus were potent and selectively inhibitory for one or two cell lines. Antiprotease activity against one or more enzymes was detected in extracts from 14 plant species, with most activity being directed against leucine aminopeptidase (LAP). The activities observed are discussed in relation to the chemical constituents reportedly isolated from these plants and/or other species of the genus and their traditional uses.
Introduction
Several useful anticancer drugs, such as taxol, vinblastine, irinotecan, topotecan, etoposide, and palclitaxel, have been derived from plants (Cragg & Newman, Citation1999; da Rocha et al., Citation2001), and there is good reason to believe that many more are yet to be discovered. Fewer than 1% of tropical plants have been evaluated in terms of their medicinal potential, although 25% of prescriptions in Western countries contain components derived from plants (Farnsworth, Citation1994; Pezzuto, Citation1997).
Anticancer drugs derived from plants may act through a variety of mechanisms including a direct effect on tumor cell growth. This may be achieved through interference with the cell growth cycle or through a direct cytotoxic effect. Inducing cell death through apoptosis stands as an important approach in the search for new neoplasic drugs (Ferreira et al., Citation2002). The terpenoid Taxol (paclitaxel), obtained from Taxus brevifolia., is known to induce tumor cell death via apoptosis (Goldberg & Eisen, Citation1990; Gibb et al., Citation1997) and is one of several plant-derived anticancer drugs, which also include vincristine (Oncovin), vinorelbine (Navelbine), and teniposide (Vumon), which act directly on tumor cells (Pezzuto, Citation1997).
However, there is also at present interest in controlling tumor growth in other ways. Anti-angiogenic drugs may limit growth of tumors by restricting their blood supply; immunomodulatory agents serve to stimulate an existing antitumor immune response; and antiproteolytic drugs inhibit proteases, which are key proteins in tumor progression and metastasis, allowing cancer cell migration, angiogenesis, and vascular intravasation and extravasation (Hidalgo & Eckhardt, Citation2001).
Tumor cells release proteases at a greater rate than normal cells (Sylven, Citation1968), and the role of these enzymes at different stages of tumor cell growth has been extensively studied. Evidence exists that increased proteolytic activity contributes directly to migration and invasion by tumor cells through degradation of the extracellular matrix and basal membrane (Poole et al., Citation1978; Pietras et al., Citation1981). Different proteases appear to show a varying range of importance in different tumors. For example, several proteases have been shown to be active in colorectal carcinoma (McKerrow et al., Citation2000). In breast cancer, four classes of proteases appear to be involved in cancer progression: thiol proteases (cathepsins B and L), aspartyl proteases (cathepsin D), metal proteases (aminopeptidases, collagenases), and serine proteases (urokinase, plasmin) (Dickson et al., Citation1994aCitation1994b).
Both natural and synthetic inhibitors of metal proteases have been shown to reduce the number of colonies in a mouse model of pulmonary metastasis (Goldberg & Eisen, Citation1990). Additionally, while proteases represent an important factor in the ability of a tumor to invade a surrounding tissue, they also play a role in angiogenesis, which is, in essence, the invasion of surrounding tissues by endothelial capillary cells. Thus, primary tumors, limited by their blood supply, need to induce angiogenesis, directly or indirectly, in order to grow beyond a certain size (Folkman et al., Citation1971).
Several of these possible mechanisms of action of plant extracts on tumors are currently under study in our laboratories as part of a collaborative project to determine their possible ethnopharmacological potential in terms of their antiviral, antibacterial, analgesic, and antineoplasic activities. Venezuela is a megadiverse country with more than 20,000 species of plants so far described. Of these, indigenous and local communities use some 1500 for medicinal purposes.
The species of plants used in this study were selected on the basis of a variety of ethnopharmacological properties reported in Venezuela and other Neotropical countries. From our databases of botanical collections and ethnobiological uses, we selected 17 species to conduct an initial survey and to explore potential anticancer activity. To our knowledge, no previous studies of anticancer activity of these plant species have been reported. We present results related to their properties as potential anticancer agents, namely (a) inhibition of the growth of eight human tumor cell lines (representing lung, breast, colon and pancreas) and (b) activity against four proteases.
Materials and Methods
Study area and plant collection
All the plants were collected in the Yutaje area in northern Amazonas, a Venezuelan state, (05° 37′ 51″ N/66° 06′ 85″ W) between 1999 and 2002. Yutaje lies within a mostly uninhabited territory containing different vegetal formations including terra firme forests, low and medium altitude montane forests, flooded and riverine forests, palm swamps, and different types of savannah growing on clay, sandy, or seasonally flooded terrain. Up to now, our botanical inventory of this area reports a total of more than 4000 collections representing some 1000 species. Data of all collections are fed into a relational database system (MedPlant) using a commercial platform (FileMaker Pro). We have also set up a database containing data on Venezuelan and Neotropical plants reported to be of medicinal use in the literature (PlantMed). This database contains 11,000 files covering more than 5000 species of plants used in folk and traditional medicine in Venezuela and the Neotropics. By matching these two databases, we found that more than 280 medicinal plant species are present in the study area. From these, we selected 17 plant species of widespread use in traditional medicine to investigate potential anticancer activity (). At the time of collection for extraction, voucher samples were taken for firm botanical determination and are now deposited in Venezuelan and American herbaria. All collections are covered by legal permits obtained from the competent authority under a “Contrato de Acceso a los Recursos Genéticos” signed between IVIC and MARN (Ministerio de Ambiente y los Recursos Naturales).
Table 1. Botanical and ethnobotanical data of the plants studied.
Preparation of plant extracts
Plants were collected and extracted fresh in a field laboratory. Depending on the type of plant, different parts were collected (leaves, bark, flower, root, etc.). After grinding in a blender, plant material was macerated in three volumes of 95% ethanol for at least 3 days. The material was filtered and the alcohol extract was concentrated and dried by rotavaporation and lyophilization. Dried powder was stored at − 80 °C until use. Stock extract solutions (100 mg/ml) were prepared in 50% ethanol.
Tumor cell lines
The eight human tumor cell lines of different origins used in this study were grown and maintained in the following media: MDA-MB231 (breast) and PANC-1 (pancreas) in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS); SKBR3 (breast) and A549 (lung) in DMEM with 10% FBS and 1 mM glutamine; HT-29 (colon) in DMEM with 10% FBS and 0.45% glucose; CACO-2 (colon) in DMEM supplemented with 15% FBS, 0.005% sodium pyruvate, 0.03% glutamine, and 0.12% HEPES; CALU-6 (lung) and MCF7 (breast) in RPMI-1640 with 10% FBS and 1 mM glutamine. All cells were incubated at 37°C in a humidified 5% CO2 atmosphere.
Cytotoxicity
Tumor cells were plated at 2.5 × 104 to 5 × 104 cells/well in 100 µl of culture medium in flat-bottomed 96-well plates and allowed to attach for 24 h. Different concentrations of the extracts (10, 100, and 1000 µg/ml) in 100 µl culture medium were then added. After a further 24 h, the number of viable cells was assessed using the MTS/PMS chromogenic assay (Promega Corp., Madison, WI, USA) according to the manufacturer's instructions. Cytotoxicity is expressed as the percentage of dead cells calculated from the following formula:
where ODT0-control and ODT0-ext correspond with the measurement of optical density at time zero immediately after the addition of the reactant medium for the assay under control or extract-treated conditions, respectively. The values ODT2-control and ODT2-ext correspond with those measured after 2 h of reaction. We considered extracts to be actively cytotoxic when the value exceeded 20%.
Antiprotease activity
Leucine aminopeptidase (LAP) (EC 3.4.11.2, microsomal, from porcine kidney, Sigma, St. Louis, MO, USA) was assayed in 0.1 M phosphate buffer, pH 7.2, with 1 mM L-leucine-p.-nitroanilide (Leu-p.NA) as substrate (Cesari et al., Citation1983). Elastase (EC 3.4.21.36, from porcine pancreas; Sigma) was assayed in 0.1 M Tris-HCl buffer, pH 8.0, with 0.3 mM succinyl-Ala3-p.-nitroanilide (Bieth et al., Citation1974), and cathepsin B (EC 3.4.22.1, from human liver; Sigma) was determined using 0.2 mM CBZ-Arg-Arg-p.-nitroanilide in the presence of 5 mM L-cysteine (Barrett & Kirschke, Citation1981). In these three cases, the release of p.-nitroaniline (pNA) was measured by interpolating the absorbance values at 405 nm against a standard calibration curve of pNA. Cathepsin D (EC 3.4.23.5, from human liver; Sigma) was determined using acid-denatured hemoglobin (1.5%) as substrate in 0.1 M sodium formate pH 3.5. After incubation at 37°C for 2 h, the hydrolysis products generated by the cathepsin D action were assessed in the TCA-soluble supernatant at 280 nm and the absorbance values interpolated against a L-tryptophan calibration curve. To determine inhibitory activity, the different enzymatic assays were performed in the presence of a plant extract at a concentration of 100 µg/ml and absorbance values compared with those obtained in the absence of extract.
Results
Effect of the plant extracts on cytotoxicity in different cancer cell lines
The 40 extracts from different parts of the 17 plant species were tested at three different concentrations (10, 100, and 1000 µg/ml) on eight human tumor cell lines derived from lung (A549, CALU-6), pancreas (PANC-1), colon (HT-29, CACO-2), and breast carcinomas (SKBR3, MCF7, MDA-MB231) (). A range of sensitivities among the cell lines was observed, with SKBR3 and MDA-MB231 being the most sensitive and A549 the least. In general, most extracts not surprisingly were cytotoxic at 1000 µg/ml, the highest concentration tested, but showed a differential effect at 100 and 10 µg/ml. Thirteen extracts from 10 species show a cytotoxic effect at 100 µg/ml or less on one or more cell lines. Two extracts appeared to have a broad effect. These were the young leaf extract of Jacaranda copaia. and the extract of the inner bark of Tapirira guianensis., which were active against five and six cell lines, respectively.
Table 2. Cytotoxicity of 40 ethanolic extracts from 17 plants on 8 human tumor cell lines.
On the other hand, the twig extract of Gnetum nodiflorum. was selectively cytotoxic for two of the breast cancer cell lines (MCF7 and MDA-MB231), whereas the bark extracts of the two Protium. species, P. heptaphyllum. and P. unifoliolatum., selectively affected the two lung cancer lines at 100 µg/ml. The extracts from Euterpe precatoria, Macoubea guianensis., and Polypodium aureum. showed little activity against the cancer cell lines. However, they appeared to be cytotoxic at high concentrations (1000 µg/ml) for at least two of the three breast tumor cell lines tested, suggesting some degree of specificity for this type of cancer.
Only six extracts from four species were active at the lowest concentration tested (10 µg/ml). These were the stem extract of Costus scaber., the young leaf from Xylopia aromatica., and the young leaf and outer bark extracts of Croton cuneatus., which selectively killed the SKBR3 breast cancer line at this concentration. The young leaf and internal bark extracts of Tapirira guianensis. were cytotoxic to CACO-2 cells at this concentration.
In several cases, “negative” values for cytotoxicity were also obtained. This may correspond with the stimulation of metabolism or of cell proliferation induced by the extracts.
Antiprotease activity of plant extracts
As proteases are important in tumor growth and spread, the effect of the 40 extracts was tested on four selected proteases. Antiprotease activity against one or more enzymes was detected in 24 extracts from 14 plant species (). There was a tendency toward most antiproteolytic activity being directed against LAP with almost no effects on the activity of cathepsin B. In general, in plant species with activity against LAP, the bark extracts were more active than leaf extracts. This is the case for C. americana, C. cuneatus, E. tenuifolia, P. unifoliolatum, P. heptaphyllum, T. guianensis., and V. ferruginea..
Table 3. Effect of the plant extracts on the activity of four proteases.
Discussion
Of the 1000 species collected by us in Yutaje, more than 280 have been reported to have medicinal uses. From these, we selected 17 species widely distributed and used in the Neotropics for our studies of anticancer and other biological activities (). Most of the species tested here have not been reported in ethnobotanical surveys to be used for the treatment of tumors, although many are reported to have an anti-inflammatory action. However, other species of the same genera have been reported to show antitumor activity (Duke, Citation1998; Graham et al., Citation2000). Some of these species, or other species of the same genera, are cytotoxic for cancer cells in vitro. or contain chemical compounds with known antitumor or anti-inflammatory activity. The importance of inflammation in tumor growth and metastasis has been abundantly clear for many years (Balkwill, Citation1989), and we have shown in our laboratory that anti-inflammatory compounds may reduce the spread of metastasis in an animal model (Cubillos et al., Citation1997).
Proteases are expressed at higher levels in tumor cells (Sylven, Citation1968; Borovansky et al., Citation2000) than in normal cells and have been proposed as a possible target for tumor therapy (DeClerck & Imren, Citation1994; Kennedy, Citation1994). Many protease inhibitors, such as betulinic acid and triterpenes, are produced by microorganisms and plants in response to infection or mechanical lesions such as insect feeding and represent a defense mechanisms to such intrusions (Melzig & Bormann, Citation1998; Zang & Maizels, Citation2001; De Leo et al., Citation2002). Our results suggest that potent inhibitors of LAP are present in the ethanol bark extracts evaluated in this study. Results from our laboratory have shown that an ethanol extract of another plant, Uncaria tomentosa., inhibits both B16/BL6 melanoma cell growth in vitro. and in vivo. as well as inhibiting LAP activity in a B16/BL6 cell preparation (results not shown).
The plants, detailed below, are discussed in relation to their known chemical constituents or of other species of the genus and their uses in traditional medicine.
Costus scaber.: In our assays, we found that the stem extract of C. scaber. was highly specific and potent (at 10 µg/ml) against the SKBR3 breast cancer cell line. The extract of C. scaber. had no affect whatsoever on the activity of proteolytic enzymes. Neither the chemistry of this species nor its biological properties have been formally investigated. However, in other Costus. species, compounds with anti-inflammatory or antitumor activities have been reported. Diosgenin, from C. speciosus. (Indrayanto et al., Citation1994), has been reported to show anti-inflammatory, estrogenic, and mastogenic activities (http://www.ars-grin.gov/duke). Steroidal saponins from C. spiralis. show anti-inflammatory activity using the capillary permeability assay (da Silva & Parente, Citation2004), and flavonol diglycosides from C. spicatus. have an inhibitory activity on nitric oxide production by activated macrophages (da Silva et al., Citation2000). The compounds found in this genus may rationalize their use as anti-inflammatory and anticancer plants in traditional medicine.
Croton cuneatus.: A potent and a rather specific effect of external bark and young leaf extracts were observed on the breast cancer cell line SKBR3 at 10 µg/ml. The dichloromethane extract of the aerial parts of this species contain glutarimide alkaloids, which have been shown to be cytotoxic for the MCF7 breast cancer cell line (Suarez et al., Citation2004). In our study, we found the LAP protease to be sensitive to the bark extracts of C. cuneatus.. Other species of Croton. also display cytotoxic and antitumor activity. C. tiglium. contains isoguanosine, which has antitumor activity against implanted S-180 ascitic tumor mice and inhibits the growth of S-180 and the Ehrlich solid tumor in mice (Kim et al., Citation1994). The bark of C. malambo. contains an ent.-kaurane compound, which has cytotoxic and proapoptotic properties, inducing a decrease in BCL2 mRNA and protein and in hTERT mRNA levels in MCF7 cells (Morales et al., Citation2005). Thus, although C. cuneatus. itself is not used in traditional medicine as an antitumor plant, other species of the genus are (). C. cuneatus. may contain compounds useful in the treatment of breast cancer.
Curatella americana.: Extracts of C. americana. were not cytotoxic on the different cell lines except at high concentration (1000 µg/ml). There are no reports of cytotoxic activity of this species in the literature. The Curatella. extracts were strongly inhibitory for LAP but only mildly so for cathepsin (CAT)-D. Others have shown that a hydroalcohol extract of the bark possessed anti-inflammatory and peripheral analgesic properties (Alexandre-Moreira et al., Citation1999). The ethanol extract of the leaves contains the flavonol glycoside avicularin, as well as gallic acid (El-Azizi et al., Citation1980), both of which show anticancer activity. The strong protease inhibitory activity of this plant suggests further study of its potential anticancer activity may be merited.
Eschweilera tenuifolia.: The extracts showed inhibitory activity on three proteases (LAP, elastase, and CAT-D). We observed a potent and somewhat selective cytotoxic effect of the leaf extract on the HT-29 colon cancer cell line. Microscopy studies suggested that the leaf extract at least kills cells by necrosis rather than apoptosis (results not shown). According to current thinking, this finding would tend to eliminate it as a potential therapeutic treatment, as most researchers tend to consider an apoptotic mechanism the most desirable (Gibb et al., Citation1997; Taraphdar et al., Citation2001; Ferreira et al., Citation2002). However, given that mutations may arise in the apoptotic pathway, leading to resistance to proapoptotic drugs, it has been suggested that targeted delivery of necrosis-inducing drugs may have its advantages under certain circumstances (Kiaris & Schally, Citation1999). No biological activities or the chemistry of this species have been reported. A triterpene and the saponin sitosterol 3βO.-βD-glucopyranoside have been isolated from E. longipes. (da Costa & de Carvalho, Citation2003) as well as three ellagic acid derivatives from the bark of E. coriacea. (Yang et al., Citation1998). Saponin compounds may be responsible for the necrosis through a direct effect on the cell membrane.
Euterpe precatoria.: In general, extracts of this plant had no effect on protease activity and were not cytotoxic. E. precatoria. roots were reported to show an immunomodulatory activity through complement cascade inhibition and ADP-induced platelet aggregation inhibition assays (Deharo et al., Citation2004). No antitumor activity or use in traditional medicine has been reported in this or any species of the same genus.
Gnetum nodiflorum.: Twig extracts of G. nodiflorum. showed cytotoxic activity on the two breast cancer cell lines, MCF7 and MDA-MB231, but not on SKBR3 or any of the other cell lines studied here. Protease activity was not affected by the extracts of this plant. Plants of the genus Gnetum. contain stilbene derivatives (Tanaka et al., Citation2001; Wang et al., Citation2001; Xiang et al., Citation2002; Li et al., Citation2003Citation2004; Ohguchi et al., Citation2003) that are known to have anti-inflammatory and anticancer activities (Kimura et al., Citation1995; Ito et al., Citation2002Citation2003; Ohguchi et al., Citation2003). Another species of the genus, G. latifolium. var. funiculare., is used in traditional medicine for the treatment of cancer (Duke, Citation1998).
Jacaranda copaia.: The young leaf extract of J. copaia. showed broad cytotoxic activity against pancreas, colon, and breast cell lines, without any effect on protease activity. Of the breast cancer cell lines, SKBR3 and MDA-MB231, but not MCF7, were highly sensitive. Antitumor effects of jacaranone, a phytoquinoid, have been reported for J. caucana. (Ogura et al., Citation1976Citation1977). This species has also shown antiprotozoal activity in vitro. (Weniger et al., Citation2001). An active cyclooxygenase inhibitor, jacarandic acid, was isolated from J. mimosifolia. (Nugteren & Christ-Hazelhof, Citation1987). The dichloromethane extract of the stem of J. filicifolia. inhibits lipoxygenase in vitro. (Ali & Houghton, Citation1999). In general, members of the Bignoniaceae family contain furonaphtoquinones, which are known to be cytotoxic (Colman De Saizarbitoria et al., Citation1997). Other members of the genus Jacaranda. and of the Bignonaceae are widely used for the treatment of cancer in traditional medicine (Duke, Citation1998; Graham et al., Citation2000).
Macoubea guianensis.: M. guianensis. was not cytotoxic and had no effect on protease activity. There are no reports of cytotoxicity for this plant. The indole alkaloids, vincadifformine and vincadine, have been isolated from the seeds of M. guianensis. (Apocynaceae) (Anderson et al., Citation1985). More importantly, Catharanthus roseus., the Madagascar periwinkle, a member of the same family, is the source of the so-called vinca alkaloids, vincristine and vinblastine, drugs widely used in cancer chemotherapy.
Mandevilla scabra.: Extracts of M. scabra. inhibited LAP and CAT-B without notable cytotoxicity on cancer cell lines. Biological effects of other species of the genus have been reported. Pregnane glycoside compounds isolated from M. velutina. are effective in antagonizing BK responses and also exhibit potent and long-lasting analgesic and anti-inflammatory activities (Calixto & Yunes, Citation1991; Santos et al., Citation2003). M. illustris. contains a nor-pregnane derivative, which inhibits the rat paw edema induced by carrageenan without affecting that caused by bradykinin (Niero et al., Citation2002). There are no reports in traditional medicine of the use of plants of this genus for tumor therapy.
Polypodium aureum.: Although there are numerous reports of the antitumoral activity of several Polypodium. species, we did not find any effect on cell viability in the investigated cell lines or any antiproteolytic activity. Plants of the Polypodium. genus contain calagualine, an antitumoral saponin (Horvath et al., Citation1967), which has clinically documented medicinal uses in South America and Spain and has been shown to block tumor metastasis, proliferation, and inflammation (Manna et al., Citation2003).
Protium heptaphyllum. and Protium unifoliolatum.: The extracts of bark from these species showed cytotoxic effect on the lung cancer cell lines, A549 and CALU-6, at 100 µg/ml. This was accompanied by a potent inhibitory activity on LAP. Essential oils from species of Protium. extracted from the leaves (sesquiterpenes) and resin (monoterpenes and phenylpropanoids) exhibit anti-inflammatory activity and affect the proliferation of the neoplasic cell lines Neuro-2a (mouse neuroblastoma), SP2/0 (mouse plasmocytoma), and J774 (mouse monocytic cell line) (Siani et al., Citation1999). Also, the pentacyclic triterpenes, α.- and β.-amyrin have been isolated from the resin of P. heptaphyllum. (Susunaga et al., Citation2001; Oliveira et al., Citation2004aCitation2004b) and other Protium. species, which are associated with anti-inflammatory, antitumoral and antinociceptive activities (Mora et al., Citation2001; Otuki et al., Citation2001Citation2005aCitation2005b). However, none of the species of Protium. are used in traditional medicine for the treatment of cancer.
Psychotria poeppigiana.: None of the extracts from P. poeppigiana. showed any major effect on cell viability or protease activity. The chemistry of this plant has not been studied. However, in other members of the genus, the main bioactive compounds are neuroactive alkaloids (Carlini, Citation2003). Alkaloid extracts of P. forsteriana. show cytotoxicity on human leukemic and rat hepatoma cell lines (Adjibade et al., Citation1989). Ursolic acid, an anticancer agent, and psychorubrin, a cytotoxic naphthoquinone, have been isolated from other Psychotria. species (Hayashi et al., Citation1987; Lee et al., Citation1988).
Siparuna guianensis.: The young leaf extract of S. guianensis. showed cytotoxic activity only on the breast cancer cell line SKBR3. The extracts were without any effect on protease activity. No other species in this genus has been reported to show bioactivity against cancer cell lines or are used in traditional medicine. However, S. guianensis., as well as other species of the genus, contains benzylisoquinoline alkaloids, aporphine alkaloids, and sesquiterpenoids, compounds associated with tumor inhibitory activity (Kupchan & Altland, Citation1973; Leitao et al., Citation1999; Simas et al., Citation2001; Jenett-Siems et al., Citation2003). The cytotoxic activity on the SKBR3 cell line may be related to the presence of these compounds.
Tapirira guianensis.: The inner bark and leaf extracts of T. guianensis. were among the most broadly active of all the plants studied. Potent activity was found at 100 µg/ml against the three breast cancer cell lines, as well as CACO-2, PANC-1, and CALU-6, and also inhibited antiprotease activity. It has been reported that the aqueous ethanol extract of the dried entire plant shows cytotoxic and general toxic effects (David et al., Citation1998). The chloroform extract of seeds contains β.-sitosterol and the cytotoxic compounds, 2-[10(Z.)-heptadecenyl]-1,4-hydroquinone and (4R., 6R.)-dihydroxy-4-[10(Z.)-heptadecenyl]-2-cyclohexenone, which are active against BC1 (breast), Lu1 (lung), and Col2 (colon) cancer cell lines (David et al., Citation1998). The presence of these compounds may be responsible for the broad cytotoxicity observed. However, none of the species of the genus appear to be used in traditional medicine against cancer.
Vochysia ferruginea.: The extracts of V. ferruginea. had no effect on the viability of the cancer lines studied. However, the internal bark was a potent protease inhibitor. We have not found any reports on the chemistry of this species. None of the plants of the genus studied have shown cytotoxic activity, although they do contain cytotoxic compounds such as β.-sitosterol and betulinic acid (Hess et al., Citation1995). Vochysia divergens. is used in folk medicine against infections and asthma, and extracts and some isolated compounds from this plant have antibacterial activity.
Xylopia aromatica.: The young leaf extract of X. aromatica. exerted a cytotoxic effect against the SKBR3 and CACO-2 cell lines at low concentrations. Cytotoxic annonaceous acetogenins with activity against three human solid tumor cell lines have been isolated from the ethanol extract of the bark of X. aromatica. (Colman-Saizarbitoria et al., Citation1994aCitation1994bCitation1995). Xylopic acid and four other isolates from the fresh ripe fruits of X. aethiopica. were found to have antimicrobial properties (Boakye-Yiadom et al., Citation1977). Oxoaporphine alkaloids have been isolated from X. aethiopica., which showed selective toxicity against DNA repair and recombination deficient mutants of the yeast Saccharomyces cerevisae. (Harrigan et al., Citation1994). Extracts and essential oils from the leaves, bark, and root of X. aethiopica. were found to be very active in scavenging superoxide anion radicals (Karioti et al., Citation2004) and have antibacterial and antifungal activity (Tatsadjieu et al., Citation2003; Konning et al., Citation2004). The cytotoxic effects, the chemical compounds, and the reported usage of other species of the genus support the possible efficacy of this plant in anticancer therapy.
Conclusions
Although most of these species are not mentioned in the literature in terms of their antitumor activity, it is remarkable that in eight of the 10 species studied here with cytotoxic effect, other members of the genus have reported uses and biological activities. Most studies rely on in vitro. cytotoxicity assays to screen plants for antitumor compounds. Although several of the compounds derived from plants do act directly on tumor cells inducing death by apoptosis (Taraphdar et al., Citation2001), there is currently much interest in exploiting other mechanisms involved in tumor growth and metastasis as targets for therapy. Of 17 species, 14 had extracts with an effect on the activity of one or more of the proteases studied. However, there was not a clear relationship between the cytotoxic and antiprotease effect.
The plant species that show a cytotoxic effect on one or more cell lines, as well as those with antiprotease activity, may be the subject of further studies to determine their potential use in anticancer therapy.
Acknowledgments
We thank Giovannina Vele and Silvia Fraile for technical assistance. This work was supported by a grant in aid from Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela, and Fundación TERRAMAR S.C., Venezuela.
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