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