Cancer Letters

Cancer Letters

Volume 175, Issue 1, 10 January 2002, Pages 17-25
Cancer Letters

Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells

https://doi.org/10.1016/S0304-3835(01)00718-2Get rights and content

Abstract

Betulinic acid is a triterpene with selective cytotoxicity against melanoma, neuroectodermal and malignant brain tumor cell lines. In this study the betulinic acid activity was evaluated, in comparison with doxorubicin, on different human neoplastic and non-neoplastic cell lines and on proliferating normal lymphocytes. Growth inhibition was evident in all the neoplastic cell lines independently on p53 status and histotype. Antiproliferative activity of betulinic acid was related to a cytotoxic effect on two p53 wild-type and on one p53 mutant cell lines and to a cytostatic effect on one p53 mutant melanoma clone. At the same concentrations, normal cells were unaffected indicating a selective effect of this agent. A cytotoxic activity of doxorubicin was evident on all the tested systems. In vivo experiments, performed on one of these cell lines, confirmed the antineoplastic activity of this drug. These data support further preclinical studies of betulinic acid not confined to melanoma and neuroectodermal tumors independently of p53 status.

Introduction

Betulinic acid is a triterpene of natural origin isolated from various plants (Tryphyllum peltatum, Ancistrocladus heyneaus, Diospyros leucomelas, Tetracera boliviana, Zizyphus joazeiro, Syzygium formosanum, etc.), all widespread throughout the tropics. Betulinic acid has already shown anti-inflammatory [1], [2], antiviral [3], and antineoplastic activities [4], [5] although the clinical activity is not established.

Pisha et al. suggested that betulinic acid exerts a selective antitumor activity on human melanoma cell lines both in vitro and in vivo [5]. The authors, however, did not provide any explanation for this apparent specificity against melanoma cells. Moreover, its activity against neuroectodermal [6], [7], [8] and malignant brain tumor cells has been reported [4] and related to the induction of apoptosis via direct mitochondrial alterations [7], [8], [9], [10]. Formation of reactive oxygen species [6], modulation of BCL-2 and BAX levels [8] and topoisomerases Iα and IIα [11] have been suggested to be involved in its mechanisms of action. In melanoma and neuroectodermal tumor cells, betulinic acid activity was shown to be independent on p53 status [6], [7], [12].However, no information on the effect of this compound on normal non-transformed cells was given.

In the present study, the activity of betulinic acid was investigated both in vitro and in vivo. The in vitro cytotoxic activity was assessed in melanoma and non-melanoma tumor cell lines and was compared with that of a conventional antineoplastic drug (doxorubicin). Since a role of p53 status in the sensitivity/resistance to cytotoxic drugs has been suggested [13], [14], betulinic acid was tested on cell lines expressing a different p53 status. In vitro analysis of the differential cytotoxicity of betulinic acid in neoplastic versus proliferating normal cells was also undertaken. The in vivo therapeutic effect was assessed in human ovarian carcinoma xenografts.

Section snippets

Drugs

Betulinic acid (Sigma Chemicals, St. Louis, MO, USA) was dissolved before use at 4 mg/ml in DMSO (dimethyl sulfoxide) and diluted in RPMI 1640 containing 10% fetal calf serum (or 10% human serum in the assays performed on peripheral blood lymphocytes). The highest final concentration of DMSO was 0.75%. Doxorubicin (Pharmacia–Upjohn Co., Milan, Italy) was dissolved in saline solution and diluted in complete culture medium.

Cells

This study was performed on nine human tumor cell lines: two clones with

Antiproliferative activity

The in vitro antiproliferative activity of betulinic acid and doxorubicin against nine neoplastic and one normal cell lines was evaluated by MTT assay. As shown in Table 1, all the wild-type p53 cell lines were sensitive to doxorubicin (IC50 ranging between 0.014 and 0.063 μg/ml). Among the p53 mutant cell lines, only two (A431 and POGB) showed doxorubicin sensitivity in the same range (IC50 0.037 and 0.031 μg/ml, respectively) whereas Me665/2/21, Me665/2/60 and POGB/DX showed a reduced

Discussion

The medical treatment of many human cancers, especially those of epithelial origin, remains disappointing at present. Thus it is important to identify molecules with potential therapeutic activity, to be selected for clinical studies.

Betulinic acid has been reported to be a melanoma-specific cytotoxic agent [5]. Recently it was found to exert cytotoxic activity even on neuroectodermal [6], [7], [20] and brain tumor cells [4].

The results of this report show that betulinic acid is active in vitro

References (23)

  • E Selzer et al.

    Effects of betulinic acid alone and in combination with irradiation in human melanoma cells

    J. Invest. Dermatol.

    (2000)
  • M.L Schmidt et al.

    Betulinic acid induces apoptosis in human neuroblastoma cell lines

    Eur. J. Cancer

    (1997)
  • P.K Mukherjee et al.

    Studies on the anti-inflammatory activity of rhizomes of Nelumbo nucifera

    Planta Med.

    (1997)
  • C.M Recio et al.

    Investigations on the steroidal anti-inflammatory activity of Triterpenoids from Diospyros Leucomelas

    Planta Med.

    (1995)
  • E De Clercq

    Antiviral therapy for human immunodeficiency virus infections

    Clin. Microbiol. Rev.

    (1995)
  • S Fulda et al.

    Betulinic acid: a new cytotoxic agent against malignant brain-tumor cells

    Int. J. Cancer

    (1999)
  • E Pisha et al.

    Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis

    Nat. Med.

    (1995)
  • W Wick et al.

    Betulin acid-induced apoptosis in glioma cells: a sequential requirement for new protein synthesis, formation of reactive oxygen species, and caspase processing

    J. Pharmacol. Exp. Ther.

    (1999)
  • S Fulda et al.

    Betulin acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors

    Cancer Res.

    (1997)
  • S Fulda et al.

    Molecular ordering of apoptosis induced by anticancer drugs in neuroblastoma cells

    Cancer Res.

    (1998)
  • S Fulda et al.

    Betulin acid induces apoptosis through a direct effect on mitochondria in neuroectodermal tumors

    Med. Pediatr. Oncol.

    (2000)
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