“In vivo” antitumor activity of Larrea divaricata C.: comparison of two routes of administration

Phytomedicine, Vol. 5(1), pp. 41-45 © Gustav Fischer Verlag 1998

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"In vivo" antitumor activity of Larrea divaricata C.: comparison of two routes of administration C. Anesini, A. Genaro, G. Cremaschi, J. Boccio, M. Zubillaga, L. Sterin Borda and E. Borda Department of Pharmacology, School of Dentistry, Radioisotope Laboratory, School of Pharmacy and Biochemistry, Buenos Aires University and CEFYBO-CONICET, Buenos Aires, Argentina

Summary We have previously demonstrated that an aqueous extract from Larrea divaricata has an antiproliferative activity on T lymphoma (BW 5147) cells in culture. Moreover the extract has in vivo antitumor activity when it was administered to a pregnant rat which had a spontaneous mammary tumor. In this work, the effect of an extract of Larrea divaricata on a mammary carcinoma chemically induced with N-nitrosomethylurea in females rats was studied. The extract was administered at a dose of 250 mg/kg three times each week by two different routes, subcutaneous (s.c.) and intratumoral (i.t.). The antitumor activity of the extract was evaluated by the survival time and the percentage of tumors that decreased, remained static or increased. From tumors treated via i.t. with the extract, 7 decreased (58%), 4 remained static (33%), and 1 increased (8%); whereas from tumors treated via s.c, with the extract, 10 decreased (12%), 59 remained static (75%) and 9 increased (11 %); The total number of control tumors was 80, none of them showed regression, 12 remained static (15%) and 68 increased (85%). The survival time of treated animals using both routes (s.c, and i.t.) was significantly greater (p < 0.05) than the saline-treated rats (control): extract 70 ± 19 days, n: 32; saline (control) 38 ± 22 days, n: 20. We conclude that the aqueous extract of this plant has an in vivo antitumor activity with the intratumor route being most effective in induction of tumor regression. Key words: Larrea divaricata Cav.; antitumor activity; intratumor and subcutaneous routes.

Introduction Larrea divaricata Cav. is a plant used in folk medicine for the treatment of different deseases such as, cancer, bronchitis, fever or several inflammatory diseases (Smart et al., 1968; Martinez-Crovetto, 1981; Toursarkissian, 1980; Ratera and Ratera, 1980). In previous work (Anesini et al., 1996a) and aqueous extract from the leaves of this plant had antiproliferative activity in vitro on cells from a T lymphoma cell line (BW 5147). Also, an antiproliferative effect was obtained with the extract on concanavalin A (Con A) stimulated lymphocytes without changes in cell viability, suggesting that the extract has a suppressive but not cytotoxic effect against hyperproliferative lymphocytes. Moreover, the tumor cells were significantly more sensitive to the extract than on Can A stimulated

lymphocytes as the relation between the IC so for the extract on Con A stimulated lymphocytes and the IC so on BW 5147 cells was 20. On the other hand, the extract had no antiproliferative activity on normal lymphocytes triggering only a stimulatory effect. In attempting to elucidate the mechanism of action of the extract on BW 5147 cells, we found (Anesini et al., 1996b) that the antiproliferative activity was potentiated by NDGA suggesting the participation of leukotriene synthesis in its effects. Furthermore, the extract exerted an antiproliferative effect, increasing the levels of intracellular cAMP; but this effect was not mediated by the activation of membrane receptors associated with the adenylate cyclase system, such as ~ adrenergic and histaminergic receptors. It is interesting to note

42

C. Anesini et al.

th at when the extract was admi nistered sub cutaneously to female rats with mammary carcinomas chemic ally induced with N-nitroso-N-meth ylurea (NMU), an in vivo antitumor activity was shown. The tumors remain ed static, whereas some of them regressed. N evertheless, complete tumor regression was not observed in any tr eat ed animal. It is wo rth noting that the extra ct significa ntly increa sed th e surviva l time (Anesini et aI., 1997 ). Based on the se pr evious results, and with th e aim to evaluate the bioavailabil ity of the active compounds, a local ro ute of administr ati on, such as intratumor, wa s used in this study. The result s were compared with th ose obtained by the sub cutan eous route, on a model of chemical carcinogenesis induced with NMU in female rat s.

Materials and Methods Drugs

N -nitroso-N- methylurea was synthesized in our laborato ry and was dissolved in ph ysiologic solution before administra tio n to the rats, a fresh solutio n was pr epar ed for each injection.

Animals

• Rats: For the determinati on of th e "in vivo" antitumor activity of the extract we used Sprague-Daw ley female rats with a body weight of approximately 200 g. Th e anima ls were bred in our Institute, maintained in sta inless steel cages with cycles of 12 h of light and 12 h of dark and fed with wa ter and sta nda rd meal s ad libitum. Fifty-two rats were random ly chosen for th e experi menta l groups. Induction of mammary carcinomas

M ammary tumors in female Sprague-Dawley rat s, wer e induced by three injection s of the carcinogen Nnitro so-N-methylurea (NMU) [intraperitoneally (ip)] at a dose of 50 mg/kg, wh en the animals were 50, 80 and 110 days old (Gullino et al., 1975). Tum or sizes were mea sur ed at weekly intervals with a caliper and their growth parameters and histopathological propert ies were documented . Determination of the in ivo antitumor activity of the extract by different routes

We used for this study the leaves of Larrea div aricata Cav, (Zygophyllaceae) (Anesini and Perez, 1994 ). Thi s plant was identified by employing morphological, ana tom ical and histochemical techn iqu es in comparison with voucher specimens of herb ar ium standards. On e set of the vouchers were dep osited at our Institute and the other set at the Museum of Pharmacobotany, School of Pharmacy and Biochemi stry, University of Buenos Aires.

To determinate the in vivo antitumo r effect of th e extr act by two different routes, tu mor displa ying rat s were divided into three gro ups: 12 anim als were tr eated with the extract, using th e intra tumoral route, 20 were tr eated with th e extract sub cutaneously and 20 anima ls recived only vehicle (control group ). The extract was administered at a dose of 250 mg/kg three times weekly, begining when the tu mors appeared. Two par ameters were an alyzed to determine the antitumor activity of the extract: th e survival time, and the percentage of tumors th at increased, decreased or remain ed static as measured by tum or size variations as a functio n of time. The surviv al time was determined as the time from tumor appear ance unt il the death of the animals .

Preparation of the extract

Statistical analysis

Plant material

An extract wa s prepar ed fro m air-dried leaves as follows, to facilitate th e ext rac tion, 3 g fractions from a tot al of 120 g of th e plant were infused with 40 ml of sterilized boiling distilled water, and heated with stirring for 45 minutes at 56 °C. After cooling, each extract was macerated at 5 °C during 72 hs. The sup ernatants were taken, pooled, hom ogeni zed and dessicat ed in a ro ta vapor, giving 23 gra ms of a viscous residue . An aqueous suspension of the extract was prepared by redissolving the residue in distilled wa ter. A concentration of 1.6 g/ml wa s obtai ned an d different dilutions were mad e to obtain th e experime ntal concentrations used in thi s study. The vehicle alone wa s used as a con trol.

Differences in cumulative mor tali ties were evaluated by th e Stud ent 's T test.

II Results Determination of the in vivo antitumoral activity of the extract by different routes

To investigat e whether Larrea divaricata leaves, would exert in vivo antitumor activity on a mammar y carcinoma chemicall y induced with N -nitroso-N-meth ylurea; groups of rats were administered with an aqueous extract using two differ ent ro utes, i.e. subcutaneou s (s.c.) and intratumoral (i.t.). Fig. 1 shows the evolution

"In vivo" antitumor activity of Larrea diuar icata

c.: comparison of two routes of administration

43

B Extract subcutaneous group

A Control group

7,

Tumor (em)

Tumor (em)

7 r-

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5

L

I

J 3

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1

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40

30

20

10

Ti me (days)

o

I

I

'

I

'

10

20

30

40

50

,

60

70

80

T ime of t reatment (days)

C Extract intratumoral group Tumor (em) 7I

I

:t

J I

3

2

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ato

10

~=:+----.

20

30

40

50

Time (days) •

Treated tumor

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60

70

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80

Fig. 1. Comparative kinetic evolution of mammary tumors. Panel A: 5 tumors (0) of a rat injected subcutaneously with vehicle; panel B: 4 tumors (e) of a rat, tr eated with extract s.c. and panel C: 1 tumor (+) extract treated i.t. and 3 tumors (0) untreated of a rat. Values are repre sentative of one rat in each group . The treated rat s received 250 mg/kg of Larrea diuaricata leaf extract subcutaneously or intratumora ly, three times weekly, begining at time of tumor app earance. The size of tumors is expressed in em and the evolution was documented through time. The different signals represent different tumors of a rat. Each rat had about 4 - 5 tumors.

44

C. Anesini et al.

of mammary tumors untreated rats injected with vehicle (panel A); extract s.c. (panel B) and i.t. (panel C). It can be seen that five tumors corresponding to a control representative rat developed during the study period (0 to 40 days) with different kinetics. Either s.c. or i.t. treatment caused an antitumor effect, with most of the tumors static. Panel C shows the development of mammary tumors corresponding to a rat that has been treated i.t. only in one of them. It can be seen, that the i.t. extract treated tumor completely regressed at 55 days, and the other tumors, remained static. Data in Table 1 confirmed these observations and quantify the evolution of tumors corresponding to 20 animals treated s.c. with the extract; 12 animals treated i.t, and 20 animals with the vehicle (control). The results show the total number of tumors studied and the number of tumors that regressed, remained static or progressed in different groups. As shown in Table 1, animals treated s.c, induced a 75% stabilization and i.t. treated animals triggered a 58% regression in comparison with controls. Moreover, an increase in the survival time of about 47% in comparison with controls was also observed with s.c. and i.t, administration of the extract (Table 2).

Discussion In this report the antitumor activity of an aqueous extract of Larrea divaricata leaves administered by two different routes is reported. The extract was shown to produce an antitumor activity when administered s.c. and i.t, to female rats with

chemically induced mammary tumors. It is worth noting that the tumors in the s.c, extract-treated animals stopped growing and remained static, whereas some of them regressed. A majority of the i.t. extract-treated tumors regressed, six of them showed complete regression and only one a partial regression. The local route (i.t.) appeared to be more effective in producing tumor regression than the systemic route. This may be related to the decreased bioavailability of the active compounds in the extract by the systemic route. It is important to note that the tumors of the i.t-treated group that were not treated per se remained static, this can be related to the fact that NMU induces tumors with a great irrigation, and thus the drug may reach other adjacent tumors. The extract also significantly increased survival time, by both routes of administration. Moreover, the results are considered of sufficient interest to warrant further studies as this type of carcinoma is very similar to human mammary carcinomas (McGuire and De La Garza, 1973) and almost all tumors induced with this type of carcinogen contain at least some estrogen receptor proteins with binding characteristics similar to those found in human breast cancer (De Sombre et aI., 1976; Rivera et al., 1994). The extract appeared to be innocuous when it was administered at a dose of 250 mg/kg three times weekly to a pregnant rat with spontaneous mammary tumors, because the extract neither affected the sexual cycle of the rat nor the newborn rats (Anesini et al., 1996b). We know that the active compound(s) is (are) not protein(s) as this was shown in a pre-purification study by TCA precipitation, work is now in progress to identify the active compounds. Further in-

Table 1. Effect of the extract of Larrea divaricata on growth parameters of tumors. Group Total tumors Regression Stabilization Progression

Control 80 - (0%) 12 (15%) 68 (85%)

Intratumor

Subcutaneous 79 10 (12%) 60 (75%) 9 (11%)

Treated

Untreated

12 7 (58%) 4 (34%) 1 (8%)

32 - (0%) 27 (84%) 5 (15%)

Note: The results are expressed as percentage of tumors that decreased increased or remained static in controls and extract treated animals by subcutaneous and intratumor routes. The animals received 250 mg/kg of extract three times weekly, beginning at the day of tumor appearance. The percentages were calculated considering the total of tumors analyzed.

Table 2. Effect of the extract of Larrea divaricata on survival time. Group

Control

Subcutaneous

Survival time (days)

38

70

±

22

±

19

Intratumor 76

±

19

Note: The results are expressed in days as mean ± SD of 20 control animals, 20 extract-treated subcutaneously and 12 extract treated intratumoraly. The differences are significantly accordingly to the Student't test.

"In vivo" antitumor activity of Larrea divaricata vestigation is require to elucidate the active compounds of the extract and to determine the mechanism of action of the compounds. Acknowledgements The authors thank the Buneos Aires University for a grant (OD 014) that supported this work.

References Anesini, C. and Perez, c.: Screening of plants used in Argentine folk medicine for antimicrobial activity. ]. Ethnopharmacol. 39: 119 -128, 1994. Anesini, c., Genaro, A., Cremaschi, G., Sterin Borda, 1., Cazaux, c., Borda, E.: Immunomodulatory action of Larrea divaricata Cav., Fitoterapia, 67: 329 - 333, 1996a. Anesini, c., Genaro, A., Cremaschi, G., Zubillaga, M., Boccio, J., Sterin Borda, 1. and Borda, E.: "In vivo" and "In vitro" Antitumoral Action of "Larrea divaricata Cau". Acta Physiol. Pharmacal. Therap, Latinoam. 46 (1): 33 - 40, 1996b. Anesini, c., Boccio, J., Crernaschi, G., Genaro, A., Zubillaga, M., Sterin Borda, 1. and Borda, E.: "In Vivo" Antitumoral activity and acute toxicity of Larrea divaricata Cav. extract. Accepted for publication, Phytother, Res., 1997. De Sombre, E., Kledzik, G., Marshall, S. and Meites, J.: Estrogen and prolactin receptor concentrations in rat: mammary tumors and response to endocrine ablation. Cancer Res. 36: 354 - 358, 1976.

c.: comparison of two routes of administration

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Gullino, P., Pettigrew, H., Grantham, H.: N-nitrosomethylurea as mammary gland carcinogen in rats.]. Natl. Cancer Inst. 54: 401- 404, 1975. Martinez-Crovetto, R.: Plantas utilizadas en medicina en el Noroste de Corrientes. Ministerio de Cultura de la Naci6n, Tucuman, pp. 13 -125, 1981. Me Guire, W. and De La Garza, M.: Similarity of estrogen receptors in human and rat mammary carcinoma.j. Clin. Endocrinol. Metab. 36: 548 - 552, 1973. Ratera, E. L. and Ratera, M. 0.: Plantas de la Flora Argentina empleadas en medicina popular. Hemisferio Sur, Buenos Aires, pp. 1-105, 1980. Rivera, E., Andrade, N., Martin, G., Melito, G., Crico, G., Mohamad, N., Davio, c., Caro, R., Bergoc, R.: Induction of mammary tumors in rats by intraperitoneal injection of NMU. Cancer Lett. 86 (2): 223 - 228, 1994. Smart, c., R., Hogle, H. H., Robins, R. K., Broom, A. D. and Bartholomew, D.: An interesting observation on nordihydroguaiaretic acid (NSC 4291; NDGA) and a preliminary report, Cancer Chemother. Rept., part 1, 53: 147-151, 1968. Tousarkissian, Martin: Plantas Medicinales de la Argentina: Sus nombres botanicos, vulgares, usos y distribucion geografica, Hernisferio Sur, Buenos Aires, pp. 1-139, 1980.

Address E. S. Borda, CEFYBO-CONICET, Serrano 669, 1414, Buenos Aires, Argentina, Fax: 54-1-8562751