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Cisplatin is an efficient anticancer agent. Cisplatin-based chemotherapy is believed to involve different signal transduction pathways, among which calpain activation has been proposed as an important factor in the induced apoptosis. In our study, based on real-time single cell analysis, we investigated the molecular involvement of calpain in cisplatin-induced apoptosis in living human lung adenocarcinoma cells. After cisplatin treatment, calpain was activated, resulting in Bid cleavage at 4–5 hr, followed by Bid translocation and cytochrome c release, leading to cell death. Calpeptin and PD150606, specific inhibitors of calpain, blocked Bid activation completely; however, cytochrome c release was delayed by more than 2 hr, which was associated with the delay of caspase-3 activation and cell death. Remarkably, calpain-mediated release of cytochrome c and cell death was significantly compromised in the Bid knockdown cells. Z-IETD-fmk and Z-VDVAD-fmk were used to block the activation of caspase-8 and caspase-2, respectively; however, the progression of apoptosis were not affected, suggesting that caspase-8 and caspase-2 were not involved in this experimental model. Taken together, the data demonstrate that calpain mediated cisplatin-induced apoptosis in human lung adenocarcinoma cells through activating Bid, which then regulated the mitochondrial apoptotic pathway. The delays of cytochrome c release, caspase-3 activation and subsequent cell death by inactivating calpain or silencing Bid exclude other earlier or parallel pathways, strongly suggesting that the calpain-mediated pathway is the kinetically earliest one, which dominates the cisplatin-induced apoptosis. © 2008 Wiley-Liss, Inc.
Cisplatin (cis-diammine dichloroplatinum; cis-Platinum(II)) is a potent anticancer drug for the treatment of various malignancies.1, 2 The cytotoxicity of cisplatin results from the formation of DNA adducts, which include interstrand and intrastrand DNA cross-links, DNA-protein cross-links and DNA monoadducts.1, 2 Cisplatin is recognized as a DNA-damaging agent. Yet the mechanism whereby DNA damage kills cells is not fully understood. Inhibition of DNA synthesis was initially considered to be the main cause of cisplatin's cytotoxicity. However, recent studies demonstrate that besides inhibition of DNA replication, cisplatin treatment also affects other fundamental cellular processes, including RNA transcription, protein translation, DNA repair, cell cycle and apoptosis.1–3 Considerable evidence has indicated that cisplatin can kill cells through the induction of apoptosis. However, it is not well known how cellular signaling from drug-induced DNA lesions leads to the execution-phase characteristics of apoptosis.
Two cytosolic proteolytic systems, caspase and calpain, are capable of producing limited cleavage of endogenous proteins. Calpain is a ubiquitous cysteine protease. Although it is well established that several members of the caspase family are involved in apoptosis,4, 5 the physiological role of calpain in apoptosis is much less clear.6 Ablation of the common noncatalytic 30-kDa subunit of calpain causes embryonic lethality in mice, pointing to the essential role of the enzyme.7 A number of studies have shown that calpain activation precedes cell death induced by different apoptotic stimuli in various cell systems.8, 9 The potential role of calpain in apoptosis is also indicated by the growing list of calpain substrates, including p53, PARP, Bid and several cytoskeletal proteins.10, 11 Currently, the contribution of calpain to apoptosis is generally accepted, though its exact role remains unclear.
Two major distinct apoptosis pathways have been described for mammalian cells. One involves caspase-8, which is recruited by the adapter molecule Fas/APO-1-associated death domain protein to death receptors upon extracellular ligand binding.12, 13 The other is the mitochondrial apoptotic pathway, involving Bcl-2 family members, cytochrome c and caspase-9.14–16 Several studies have suggested that caspase-8 is involved in cisplatin-induced apoptosis.17, 18 However, Wang et al. found that cisplatin could induce apoptosis without activation of caspase-8.11, 19, 20 The debate over the involvement of caspase-8 activation in cisplatin-induced apoptosis continues.
Bid, a member of the BH3-only subgroup of the Bcl-2 family, is a unique proapoptotic protein.21 BH3-only proteins are implicated as essential regulators of apoptosis.22, 23 Bid plays an essential role in apoptotic signaling, by inducing the proapoptotic functionality of Bak and Bax, leading to cytochrome c release.24 Bid is normally localized in the cytosol in an inactive form. Activation of Bid depends on the cleavage of intact Bid into its truncated form of tBid. The resulting tBid then translocates into mitochondria and leads to disruption of organelles and release of apoptogenic molecules such as cytochrome c.13, 25, 26 The cleavage of Bid can be conducted by several proteases. Caspase-8 has been shown to be the protease most responsible for Bid cleavage during death receptor-mediated apoptosis.25 Calpain has also been shown to cleave Bid.11, 27, 28 Other studies have demonstrated that Bid can also be cleaved by caspase-3 and caspase-2 in the intrinsic pathway to apoptosis, which is independent of death receptors.29, 30
Fluorescence resonance energy transfer (FRET) is a nonradiative transfer of energy between 2 fluorophores that are placed in close vicinity and in a proper relative angular orientation, which can spatio-temporally monitor cell events in physiological condition in single living cell.31, 32 It has been utilized to reveal the dynamic activity of enzyme, and it provides a view of protein location, protein translocation, small ligand binding, protein–protein interaction, conformational change and posttranslational modification in real-time.33–36 This can not be fully elucidated by traditional biophysical or biochemical approaches, which can only measure the average behavior of cell populations and the static spatial information available from fixed cells and thus can not provide direct access to cells life event in their natural environment.
Given these backgrounds, it is of importance to determine the effect of calpain activation in cell apoptosis, particularly induced by chemotherapeutic agent such as cisplatin. To determine the molecular involvement of calpain in cisplatin-induced apoptosis in living human lung adenocarcinoma cells, our study focuses on (i) calpain activation and its contribution in the apoptotic pathway, (ii) the dynamic process of cell apoptosis, including Bid activation, Bid translocation, cytochrome c release, (iii) the role of caspase-2, -3 and -8 in cell apoptosis and (iv) the dominant apoptotic pathway in this experimental model.
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Many reports have shown that apoptosis is a marker of tumor cells exposed to cisplatin.1, 2 However, the mechanisms of cisplatin-mediated apoptosis are not fully understood. Considerable evidence has indicated that factors such as p53, calpain, Bcl-2 family, caspases, CD95 and IAPs may influence the ability of cisplatin to induce apoptosis.2, 11, 42–44
Possible involvement of calpain in apoptosis was first suggested in 1993.45 Involvement of calpain has been observed in response to irradiation, serum deprivation, staurosporine, etoposide and cisplatin.2, 9, 11, 44 These studies suggest that calpain-mediated cleavage of Bcl-2 family proteins (Bax, Bid, Bcl-2 and Bcl-xL) results in conformational changes, hence their activation.11, 27, 46
The current study addresses several essential issues of cisplatin-induced tumor cell apoptosis, including the dynamics of apoptotic process and the roles of calpain and caspases.
Apoptosis induced by cisplatin in human lung adenocarcinoma cells was studied using real-time single-cell analysis. Fluorescence images of tumor cells transfected by GFP (Fig. 1a, 2nd panel) clearly show cell apoptosis induced by cisplatin. After cisplatin treatment, calpain was activated (Fig. 2a), resulting in Bid cleavage at 4–5 hr, evidenced by the decrease of FRET [Figs. 3a (2nd panel) and 3b], followed by Bid translocation (Figs. 4b, 4f and 4g), cytochrome c release (Figs. 5b and 5e) and caspase-3 activation (Fig. 2d), leading to cell death.
To investigate the role of calpain activation in the induced apoptosis, cisplatin was coadministered with calpeptin or PD150606. Bid cleavage was blocked by the use of calpeptin, as shown by the stable FRET in Figures 3a (3rd panel), 3c and 3d. Figures 4b, 4d and 4e vividly show the dynamics of Bid translocation to mitochondria, caused by the treatment of cisplatin, while images in Figure 4c show undisturbed mitochondria in cells cotreated by cisplatin and calpeptin. Furthermore, the cytosolic GFP-Cyt-c fluorescence images of cells indicate that the use of calpeptin or PD150606 did not block cytochrome c release (Figs. 5c and 5d), but only delayed its onset (Figs. 5c, 5d and 5e), hence delaying caspase-3 activation (Fig. 2d) and cell death (Fig. 1). Our data demonstrated that Bid is the critical substrate of calpain in the study model, knock-down of Bid had the same effect on delaying cytochrome c release and cell death compared to the calpeptin treatment (Figs. 6c and 6d). These results suggest that cisplatin activates other signals in addition to the calpain-mediated pathway. Previous studies have shown that p53 is a mediator in cisplatin-induced apoptotic pathways,1, 2, 43, 47 which can initiate apoptosis by transcriptionally activating proapoptotic Bcl-2 family members (e.g., Bax, Bak) or directly activating Bax. It is found that cisplatin-induced activation of the ER-specific caspase-12 resulted in apoptosis.44 Other studies have demonstrated that extracellular signal-regulated kinases (ERK) can also be activated by cisplatin treatment in Hela cells.19 On the basis of our findings, the dominant mechanism of cisplatin-induced cell death is the calpain-mediated Bid cleavage and translocation. As for the delayed cell death after calpain is inhibited, the mechanism is not clear. We are currently investigating the additional apoptosis mechanism not requiring the Bid activities, such as p53-mediated apoptotic pathway.
The pathways involved in the cross-talk between the calpain and caspase proteolytic system remain controversial.9, 48–51 For example, calpain-mediated N-terminal truncation of caspase-3 to a p30 polypeptide enhanced caspase-3 activation in one study52 and inhibited its activation in another,53 while Dursun et al. reported that caspases and calpain are independent mediators of cisplatin-induced endothelial cell necrosis.54 In current study, the calpain inhibitors Calpeptin and PD150606 was found to obviously delay the activation of caspase-3 in cisplatin-induced apoptosis, which was associated with the delay of cytochrome c release and cell death, suggesting that calpain activation proceeds the activation of caspase-3. Previous studies reported that caspase-3 can cleave the calpastatin, an endogenous calpain inhibitor, leading to loss of the inhibitory effect of calpastatin and to increase of calpain activation.48, 55, 56 However, in our study, calpain activity, which was inhibited significantly with calpeptin and PD150606, was unaffected by the caspase-3 specific inhibitor (Fig. 2a), suggesting that caspase-3 had no contribution to calpain activation.
Caspase-8 has been shown to be the protease most responsible for Bid cleavage during death receptor-mediated apoptosis.13, 25 However, the involvement of caspase-8 in cisplatin-induced apoptosis remains highly debated.11, 17–20 We used caspase-8 specific inhibitor Z-IETD-fmk with cisplatin in the treatment of tumor cells. Comparing the fluorescence images in Figure 1a (2nd and 5th panel), it is evident that Z-IETD-fmk has no effect on apoptosis induced by cisplatin. The measured FRET (Fig. 3a, 4th panel), YFP and CFP emissions (Figs. 3c and 3d), western blotting analysis (Fig. 3e), as well as cellular distribution of Bid-CFP (Fig. 4d) further indicate that Z-IETD-fmk had no effect in Bid cleavage and Bid redistribution. Western blotting experiments and the enzymatic assay for caspase-8 activity also demonstrated that caspase-8 was not activated (Figs. 2b and 2c). Therefore, our results excluded caspases-8 from having any important role in cisplatin-induced apoptosis.
Interestingly, Z-VAD-fmk showed no major effects on Bid cleavage and translocation (Figs. 3c, 3d and 4e), but only slightly slowed the process of Bid-CFP translocation to mitochondria (Fig. 4f). The data suggest that Z-VAD-fmk is not a specific caspase inhibitor and can slightly inhibit calpain as shown in Figure 2a, which is consistent with the observations of previous studies.9 Further studies are needed to elucidate the mechanism.
Despite considerable evidence indicating that caspase-2 is activated early in response to a variety of apoptotic stimuli, assigning a distinct function to this protease has been difficult. Recent findings indicate that caspase-2 is an important initiator of the mitochondrial apoptosis pathway, which is required for the permeabilization of mitochondria and release of cytochrome c and Smac/DIABLO.38–41 Considering the number of pathways linking cytotoxic stress with mitochondrial engagement, it seems likely that the extent of caspase-2 involvement depends on a number of factors, including the nature of the cytotoxic stimulus, the cell type that is being affected and the intracellular abundance of caspase-2 relative to that of other proapoptotic proteins. Indeed, experiments using caspase-2 knockout mice, or cells in which caspase-2 had been downregulated by antisense and siRNA approaches, as well as studies with caspase-2 inhibitors, suggested that caspase-2 activation seems to be important for cell death in many, but not all, cell types.38–41 The results in our current study demonstrate that caspase-2 was not involved in the progression of cisplatin-induced apoptosis in human lung adenocarcinoma cells [Figs. 1a (6th panel), 1b and 7], indicating that the role of caspase-2 in apoptosis is cell- and trigger-specific.
Bid plays an essential role in apoptosis. It has been established that Bid serves the unique function of interconnecting the extrinsic death receptors for TNF-α and Fas to the mitochondrial amplification loop of the intrinsic pathway.13, 25 After TNF-α or Fas treatment, Bid is cleaved by caspase-8 at Asp59 after the LQTD site, yielding a p15 C-terminal truncated fragment and exposing a new amino terminal Gly residue, which becomes myristoylated, facilitating Bid translocation to the mitochondria, where it induces the activation of Bax and Bak, resulting in the release of cytochrome c.13, 25, 26, 57 Therefore, the cleavage and redistribution of Bid has a profound effect on proapoptotic activity. Our data demonstrated that cleavage of Bid by calpain modulate cisplatin-induced apoptosis. However, it was reported that Bid was cleaved by calpain at Gly70 after the SRLG site, generating a p14 tBid and exposing an Arg residue,11 which is different from that cleaved by caspase-8. Therefore, posttranslational N-myristoylation of Bid may not explain the mechanism by which Bid translocates to mitochondria in the cisplatin-induced apoptotic pathway. How Bid rapidly and selectively targets the mitochondrial outer membrane should be further explored. It is possible that Bid can be activated by other forms of posttranslational modification such as phosphorylation. Alternatively, Bid may be regulated by proteins such as 14-3-3, which regulate the activity of another BH3-containing protein, Bad.58
According to our experimental data and related analysis, we demonstrated that cytochrome c release and subsequent cell death caused by calpain activation appears to be the earliest action in cisplatin-induced apoptosis, as evidenced by the delay of cytochrome c release (Figs. 5c, 5d and 5e) and subsequent delay of caspase-3 activation (Fig. 2d) and cell death (Fig. 1) by the inhibitory effect of calpeptin or PD150606 or by knock-down of Bid (Figs. 6c and 6d). Although detailed information on other pathways is not fully available, the delay of cytochrome c release and subsequent delay of caspase-3 activation and cell death by the inactivation of calpain exclude other earlier or parallel pathways, hence establishing the dominance of the calpain-mediated apoptotic pathway.
In conclusion, we demonstrated that calpain plays an important role in the cisplatin-induced apoptotic pathway. More importantly, our findings suggest that the calpain-mediated pathway is the kinetically earliest pathway, which may dominate the cisplatin-induced apoptosis. Further studies are needed to determine how the calpain-mediated pathway interacts with other signal pathways. Our findings contribute to an improved understanding of the mechanisms involved in proapoptotic signaling mediated by cisplatin-based chemotherapy.