Maslinic acid, a natural triterpene from Olea europaea L., induces apoptosis in HT29 human colon-cancer cells via the mitochondrial apoptotic pathway
Introduction
In recent years intensive efforts have been made to identify new natural antitumoral agents. Triterpenoids are compounds present in a wide range of plants used in traditional medicine and known to have antitumoral properties [1], [2], [3]. Low concentrations of maslinic acid (Fig. 1) are to be found in plants with medicinal properties, such as Cornus kousa, Junillia aspera, Malus pumila, and Ulmus pumila [4], [5], [6], [7] but its concentration in the waxy skin of Olea europaea L. may be as high as 80%. It is known for its important effects as an anti-oxidant [4], [8], [9], an anti-inflammatory [10] and its anti-microbial and anti-viral activities [11], [12], [13]. The identification of new cytotoxic agents that enhance or restore the capability of malignant tumour cells to undergo apoptosis may be crucial for more effective anticancer therapies. Triterpene compounds have been reported to be effective against some drug-resistant cells [14], [15], [16]. Nevertheless, the mechanism for the antitumoral activity of maslinic acid has not yet been explored. The normal process of apoptosis is inactive in several cancer cells lines but in many cases natural compounds can restore this process through two major pathways: the intrinsic and the extrinsic [17], [18], [19].
One key mechanism in the process involved in the function of antitumoral drugs is the activation of the mitochondrial apoptotic pathway. The mitochondrial function via the intrinsic apoptotic pathway (mitochondrial pathway) appears to be controlled by the Bcl-2 family of proteins [19], [20]. The inhibition of Bcl-2, an anti-apoptotic protein of the Bcl-2 family, and the activation of Bax, a pro-apoptotic protein of the Bcl-2 family, result in mitochondrial disruption and the release of pro-apoptotic mitochondrial factors such as cytochrome-c. When released cytochrome-c interacts with Apaf-1 and activates caspase-9, which in turn proteolytically activates caspase-3 downstream; this caspase is the principal protease in the induction phase of apoptosis. We show here that maslinic acid induces cell death through the direct activation of the mitochondrial apoptotic pathway.
Bcl-2 protein is over-expressed in many types of cancer cell. It blocks apoptotic cell death and protects the disruption of mitochondrial cells. Other triterpenoids such as betulinic acid have been shown to induce apoptosis by activating this pathway, although the over-expression of Bcl-2 tends to block the apoptosis produced by this compound. Bcl-2 inhibition has also been observed in the CDDO and CDDO-Me, in which case a loss of mitochondrial membrane potential and Bax activation has been found [16], [21]. Finally, It has been reported that other oleanane derivatives such as oleanolic acid are responsible for a wide variety of antitumoral effects in vivo, including the inhibition of angiogenesis [22] and tumour promotion [15], but the molecular mechanisms by which natural oleananes promote these processes have not yet been fully identified.
Anti-tumoral effects of maslinic acid have been reported previously in CaCo2 and HT29 colon-cancer cell lines [23], [24] and in the astrocytome cell line [25]. A loss of mitochondrial membrane potential has also been described in response to maslinic-acid treatment [23], [25]. Kim et al. have reported the cytotoxic effect of maslinic acid on the following tumour cell lines: A549 (non-small lung cells), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF-498 (central nervous system), and HCT-15 (colon) [26]. The detailed molecular mechanism by which maslinic acid induces its apoptotic and cytotoxic effects has not, however, been reported before this present study.
We have evidence to show that maslinic acid exerts anti-proliferative and pro-apoptotic effects in the HT29 colon adenocarcinoma cell line. It also induces morphological changes that are characteristic of apoptosis, such as chromatin condensation and fragmentation, as well as cell shrinkage [23]. We describe in this paper the morphological apoptotic changes and percentage of apoptotic cells as determined by fluorescence microscopy, and propose a possible molecular mechanism to explain these processes. Our results suggest that maslinic acid acts by directly inhibiting the expression of Bcl-2, increasing that of Bax, releasing cytochrome-c from the mitochondria and activating caspase-9 and then caspase-3. We report for the first time the precise molecular mechanism by which maslinic acid produces its apoptotic effects in HT29 colon carcinoma cells. Natural agents such as maslinic acid that are able to activate the intrinsic process of programmed cell death may prove to be a useful approach both to the chemoprevention and chemotherapy of cancer. Thus, maslinic acid isolated from olive pomace may provide a useful new therapeutic strategy for the treatment of colon carcinoma.
Section snippets
Materials
Dulbecco’s modified Eagle’s medium (DMEM, Sigma, St. Louis, MO, USA), foetal calf serum (FCS, Gibco-BRL, Eggenstein, Germany), penicillin/streptomycin (Gibco-BRL, Eggenstein, Germany), phosphate buffered saline (PBS, Sigma, St. Louis, MO, USA), mowiol (Calbiochem, Merck, Darmstadt, Germany), annexin V-FITC (Bender Med-Systems, Vienna, Austria), propidium iodide (PI, Sigma, St. Louis, MO, USA), culture flasks, and well-plates (Techno Plastic Products, Trasadingen, Switzerland).
Drugs
Maslinic acid, a
Effects of maslinic acid on HT29 colon-cancer cell proliferation
In a previous work [23], we evaluated the differentiation-inducing activity and the selective antitumoral effect of maslinic acid, a newly discovered triterpenoid isolated as the main compound from olive-skin pomace. In this study we have evaluated the effects of maslinic acid on cell proliferation (Fig. 2) and apoptosis in HT29 colon-cancer cells. In this cell line the concentration of maslinic acid required for 50% growth inhibition (IC50) was 28.8 ± 0.9 μg/mL and the concentration required for
Discussion
Chemopreventive agents of a natural origin, often a part of our daily diet, may provide a cheap, effective way of controlling such diseases as cancer of the colon. A wide range of studies in recent years has shown that triterpenoids hinder carcinogenesis by intervening in pathways such as carcinogen activation, DNA repair, cell cycle arrest, cell differentiation and the induction of apoptosis in cancer cells. This study is the first to investigate the molecular mechanisms of the anti-tumoral
Conflicts of interest statement
I, José Antonio Lupiáñez Cara, declare that: I have no proprietary, financial, professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in the manuscript entitled.
Acknowledgements
This study was supported by Grants AGL2000-1534, AGL2006-12210-C03-02/ALI, SAF2005-01627, ISCIII-RTICC (RD06/0020/0046) from the Spanish government and the European Union FEDER funds. We thank to Dr. Andres Garcia-Granados for providing the maslinic acid. We also thank our colleague Dr. J. Trout for revising our English text.
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2023, PhytomedicineCitation Excerpt :In 2021 it was suggested that MA increased the HT-29 cellular susceptibility to apoptosis through the NF-κB pathway by inhibiting inhibitor of nuclear factor kappa-B kinase subunit beta (IKK-β) activation (Ooi et al., 2021). In Caco-2 and HCT 116 cells, the genotoxicity or the apoptotic event could be detected in 24 h or earlier after MA treatment (Reyes-Zurita et al., 2016). However, in HT-29 cells late induction of apoptosis (72 and 96 h) was observed (Reyes-Zurita et al., 2011; Ooi et al., 2021).