Caspase-3-dependent protein kinase C delta activity is required for the progression of Ginsenoside-Rh2-induced apoptosis in SK-HEP-1 cells

Abstract

Ginsenoside-Rh2 (G-Rh2) has been shown to induce apoptosis in a variety of cell types. In this study, we show that G-Rh2-induced apoptosis is accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in the human hepatoma cell line, SK-HEP-1. Furthermore, protein kinase C delta (PKCδ) activity was markedly up-regulated in a lipid activator-independent manner with kinetics similar to those of PKCδ and PARP cleavages during the apoptotic progression. Pre-treatment of cells with the caspase-3 specific inhibitor (z-DEVD-fmk) effectively prevented the G-Rh2-induced proteolytic activation of PKCδ. Moreover, rottlerin, a specific PKCδ inhibitor blocked G-Rh2-induced proapoptotic effects on the cells including the release of cytochrome c, activation of caspase-3 activity, and proteolytic cleavage and activation of PKCδ. These results suggest that G-Rh2-induced apoptosis is functionally linked to mitochondrial dysfunction and caspase-3 activity is regulated by positive feedback with PKCδ via the mitochondrial pathway.

Introduction

Apoptosis, or programmed cell death, is a physiological process of cell elimination that not only is essential for normal tissue homeostasis, but also is critical in disease states [1]. Apoptosis is characterized by distinct morphological changes such as membrane blebbing, nuclear condensation, and fragmentation of genomic DNA [2]. Most of these morphological changes are conducted by caspases, a family of aspartate-specific cysteine proteases, which play essential roles in apoptotic cells [3]. Elimination of caspase activity retards, or even prevents, apoptosis [4].

In mammalian cells, apoptosis is initiated primarily by two pathways. One involves ligation of transmembrane death receptors such as Fas and TNFα receptors by their specific ligands [5], [6]. This stimulation results in the recruitment and activation of caspase-8, which activates downstream effector caspases such as caspases-3, -6 and -7 [7]. The other pathway depends on mitochondrial dysfunction [8]. Depolarization of the mitochondria leads to the release of cytochrome c which binds to Apaf-1 to recruit and activate pro-caspase-9, leading eventually to the activation of downstream caspases [9], [10].

In earlier reports, it has been shown that protein kinase Cδ (PKCδ) is a substrate of caspase-3 and, consequently, the kinase becomes constitutively activated in the progression of apoptosis [11], [12], [13]. PKCs comprise a family of Serine/Threonine protein kinases which are involved in intracellular signaling to regulate growth, differentiation and apoptosis [14]. There are 12 known isoenzymes of PKC that are classified into three categories: the conventional PKCs (cPKCs: α, β1, β2, γ), which are calcium dependently activated in the presence of diacylglycerol (DAG) or 12-O-tetradecanoylphorbol-13-acetate (TPA), the novel PKCs (nPKCs: δ, ε, θ, η), which are calcium independently activated by DAG or TPA, and the atypical PKCs (aPKCs: ζ, λ/ι), which are calcium-independent and not activated by DAG or TPA.

The individual roles of PKCs in the regulation of apoptosis have been reported in various systems. In most systems, PKCα [15], [16], ε [17] and ι [18] act as anti-apoptotic kinases, whereas PKCθ [19], μ [20] and δ [21] act as pro-apoptotic kinases. PKCδ is activated and translocated to the plasma membrane in a number of cell types by various apoptotic stimuli, including H₂O₂ [22], etoposide [23], [24], ionizing radiation [25], Ara-C [26], TNFα [27], UV irradiation [28], and ceramide [29]. Additionally, it has been shown that PKCδ is cleaved in the third variable (V3) region into a catalytically active fragment by caspase-3 in response to apoptotic stimuli, such as ionizing radiation [25], DNA damaging drugs [12], [20], and oxidative stress [11]. Furthermore, overexpression of the catalytic fragment of PKCδ potentiates apoptosis induced by various apoptotic agents [11], [23]. However, other studies have shown that proteolytic cleavage of PKCδ is not required for its activation during apoptosis in CHO cells treated with H₂O₂ [30], LNCap cells treated with phorbol esters [31], or HaCaT cells stimulated by UV radiation [32].

Ginsenoside-Rh2 (G-Rh2) is a diol-type ginseng saponin with a dammarane skeleton that is isolated from the root of Panax ginseng, C.A. Meyer [33]. It has been shown that G-Rh2 inhibits cell growth in MCF-7 human breast cancer cells [50] and SK-HEP-1 human hepatoma cells [37], and this agent is also capable of inducing apoptosis in various cells including C6 rat glioma cells [34], SK-N-BE(2) human neuroblastoma cells [35], and A375-S2 human malignant melanoma cells [36]. G-Rh2 rapidly up-regulates JNK1 activity [38] and induces proteolytic activation of pro-caspase-3 during apoptotic progression [39]. In SK-N-BE(2) cells, activity of PKC subtypes α, β, γ and δ was progressively increased by G-Rh2 treatment, while PKCε was gradually down-regulated. But, in C6Bu-1 cells, no significant changes in PKC subtypes were observed, suggesting that G-Rh2 may induce apoptosis via different pathways in different cell types [35]. However, the underlying mechanism by which G-Rh2 induces apoptosis is not clearly understood yet.

In this study, we investigated the involvement of PKCδ in G-Rh2 induced apoptosis in SK-HEP-1 cells. Here, we show that PKCδ is proteolytically activated by caspase-3 during G-Rh2-induced apoptosis, while other Serine/Threonine kinase activities, such as PKA or PKCα, remain unaltered. Furthermore, inhibition of G-Rh2-induced PKCδ activity efficiently prevents release of cytochrome c from mitochondria and also prevents activation of caspase-3. Thus, we suggest that caspase-3 and PKCδ form a positive feedback loop for their own activation, via the mitochondrial pathway during G-Rh2-induced apoptosis.