Asiatic acid, a triterpene, induces apoptosis through intracellular Ca²⁺ release and enhanced expression of p53 in HepG2 human hepatoma cells

Abstract

Asiatic acid (AA), a triterpene, decreased viability and induced apoptosis of HepG2 human hepatoma cells in a dose-dependent manner. AA also markedly increased intracellular Ca²⁺ level, which was blocked by TMB-8 and dantrolene, intracellular Ca²⁺ release blockers, but not by EGTA, an extracellular Ca²⁺ chelator. Moreover, AA-induced apoptosis was significantly suppressed by treatment with TMB-8 and dantrolene, suggesting that intracellular Ca²⁺ release may play an essential role in the AA-induced apoptosis. In addition, AA profoundly increased protein level of p53, which was also inhibited by BAPTA/AM, an intracellular Ca²⁺ chelator, TMB-8 and dantrolene. Treatment with A23187, a Ca²⁺ ionophore, or thapsigargin, a Ca²⁺-ATPase inhibitor, alone enhanced p53 nuclear accumulation, indicating that p53 accumulation is dependent on intracellular Ca²⁺ increase. Furthermore, the viability of Hep3B, p53-null cells, was much higher than that of HepG2, p53-wild type cells, when treated with AA. Taken together, these results suggest that AA induced apoptosis through increased intracellular Ca²⁺, which, in turn, enhanced p53 expression in HepG2 cells. These results further suggest that AA may be a valuable agent for the therapeutic intervention of human hepatomas.

Introduction

Triterpenes are the major constituent of some medicinal herbs and are widely present in all parts of a variety of plants. Triterpenes such as glycyrrhizin [1], oleanolic acid [2], ursolic acid [3], β-boswellic acid [4], have been reported to possess a variety of biological effects [5] including anti-inflammation, hepatoprotection and immunomodulation. In particular, ursolic acid has been known to have anti-tumor effects, such as skin-tumor prevention [6], inhibition of tumor promotion [7], anti-invasion [8] and induction of cancer cell apoptosis [9]. Asiatic acid (AA), a pentacyclic triterpene found in Centella asiatica, has been shown to possess an anti-ulcer effect and to protect β-amyloid-induced neurotoxicity [10]. However, to our knowledge, an anti-tumor activity of AA has not been reported yet.

Apoptosis is a highly organized cell death process characterized by loss of plasma membrane phospholipid asymmetry, enzymatic cleavage of the DNA into oligonucleosomal fragments, and segmentation of the cells into membrane-bound apoptotic bodies [11]. Apoptosis has been recognized to play an important role in maintenance of tissue homeostasis by the selective elimination of excessive cells [12]. Genetic changes resulting in loss of apoptosis or derangement of apoptosis-signaling pathways are likely to be critical components of carcinogenesis [13], [14]. Additionally, induction of apoptosis of cancer cells is recognized as a valuable tool for cancer treatment [15].

Intracellular Ca²⁺ seems to be essentially involved in the mechanism of apoptosis [16]. One of the targets for elevated intracellular Ca²⁺ is the activation of the Ca²⁺-dependent protein kinases and phosphatases [17], which has been seen during apoptosis [18]. Direct activation of the Ca²⁺-dependent proteinase may represent the other target for intracellular Ca²⁺ action in apoptosis [19]. Ca²⁺/Mg²⁺-dependent endonuclease of which action results in DNA fragmentation, the most characteristic biochemical feature of apoptosis [20] and Ca²⁺-dependent transglutaminase which is highly activated in apoptotic cells [21], also appear to be targets for Ca²⁺ action [22]. Interestingly, disruption of intracellular Ca²⁺ signal by inhibiting sarcoplasmic reticulum Ca²⁺ pumps has been shown to increase cell cycle regulator, p53 [23].

The accumulation of p53 in response to various stresses such as DNA damage, hypoxia and oncogenic signals has been implicated in growth arrest, differentiation, or senescence due to cell cycle arrest and apoptosis [24]. The downstream signaling pathways involved in p53-induced apoptosis are either transcription-dependent expressing cell death genes such as bax, IGF-BP3, and Fas/Apo-1 [25], or transcription-independent [26]. However, the upstream signaling pathways of p53 activation are essentially unknown.

Thus, in the present study, we investigated whether: (i) AA induces apoptosis in HepG2 human hepatoma cells; (ii) intracellular Ca²⁺ increase mediates the AA-induced apoptosis; (iii) there are any changes in p53 expression during apoptosis induced by AA; and (iv) increased intracellular Ca²⁺ mediates enhanced p53 expression.