Background
Cancer is an aberrant net accumulation of atypical cells, which can arise from an excess of proliferation, an insufficiency of apoptosis, or a combination of the two [
1]. The frequency of apoptosis could contribute to cell loss in tumours and promote tumour regression. Thus, in cancer therapy, the focus is on strategies that suppress tumour growth by activating the apoptotic program in the cell [
2]. Evidence accumulated to this date has established that many agents of cancer chemotherapy affect tumour cell killing through launching the mechanisms of apoptosis [
3]. Manifestations of apoptosis are easily discernible by the appearance of cell shrinkage, membrane blebbing, chromatin condensation, DNA cleavage, and finally, fragmentation of the cell into membrane-bound apoptotic bodies [
4].
Expressed as inactive proenzymes, caspases are members of a family of cysteine proteases that play a central role in the apoptotic pathway [
5]. Two major mechanisms exist that initiate the caspase cascade: the extrinsic, involving caspase-8; and the intrinsic pathway, involving caspase-9 as the apical caspase. Observations from several studies have suggested that a caspase-8 pathway can be up-regulated after drug treatment, and these include the drugs cisplatin [
6], etoposide [
7], doxorubicin and methothrexate [
8]. Once activated, caspase-8 is thought to activate the downstream caspases by proteolytic cleavage of their zymogen forms [
9,
10], thus amplifying the caspase signal. The other initiator caspase, caspase-9, controls the apoptotic response to lethal cellular insults such as ionizing radiation or certain chemotherapeutic drugs [
11]. In many systems, release of cytochrome
c from the mitochondria to cytosol has been demonstrated to be a crucial step in the activation of apoptosis [
12‐
14]. Once released from mitochondria, cytochrome
c acts as a co-factor and interacts with Apaf-1 and procaspase-9, which in turn activates caspase-9 [
15].
The role of active caspase-8 and -9 is to generate the active forms of downstream executioner caspases, including caspase-3 and -7, by limited proteolysis, and thereby transmit the apoptotic signal to the execution phase. Activation of these executioner caspases during apoptosis results in the cleavage of critical cellular substrates, thus disabling critical homeostatic and repair enzymes as well as key structural components that culminate in cell death [
16,
17].
Styrylpyrone derivative (SPD) is a pharmacologically active compound extracted from the plant
Goniothalamus sp. of the Annonaceae family [
18]. Among the species of
Goniothalamus are
G. umbrosus,
G. andersonii,
G. macrophyllus and
G. malayanus. Previous studies on SPD suggest this bioactive compound as an antiproliferative and selective cytotoxic agent.
In vitro, SPD was found to selectively inhibit the proliferation of several cancer cell lines without being significantly cytotoxic towards non-malignant cells [
19‐
21]. On
in vivo models, SPD is reported to be capable of tumoricidal and tumoristatic effects on experimental rats with mammary tumours [
22]. Recent work done to elucidate SPD's mechanism of action found evidence that SPD modulates the gene expression of
Bcl-2 and
Bax in ovarian carcinoma [
20]. In breast cancer cells, SPD induces an increase of the proapoptotic Bax protein, culminating in cell death by apoptosis [
21].
In this study, we further demonstrate the mechanism of apoptosis induced by SPD. We show that procaspase-8 was not activated in MCF-7 cells but caspase-9 activation was detected in response to SPD treatment, with the release of cytochrome c into the cytosol. This was followed by the activation of the executioner caspase-7. To further examine the involvement of this executioner caspase, we found that caspase-7 activity decreased and apoptosis was abrogated when SPD-treated cells were preincubated with the caspase-7 inhibitor, Ac-DEVD-CHO, suggesting a caspase-7-dependent apoptotic pathway induced by SPD.
Discussion
There is an increasing realization that chemotherapeutic agents act primarily by inducing cancer cell death through the mechanisms of apoptosis [
35]. However, there are many cancers that are intrinsically resistant to apoptosis, making it vital to develop novel drugs for combination chemotherapy. In the present study, we provide evidence that a compound of plant-origin, SPD, may be a promising new anticancer agent for human breast cancers.
Previously, we have shown that MCF-7 cells treated with SPD displayed elevated levels of apoptosis and a marked increase in the expression of the proapoptotic Bax protein [
21]. In addition to the loss of viability, Bax expression produces other typical manifestations leading to apoptosis, namely caspase activation [
5]. Here, we found that caspase-9 was activated, together with accumulation of cytochrome
c in the cytosol. Caspase-9 can activate downstream executioner caspases including caspase-7, which is termed caspase-3-like due to its similarity in specificity with caspase-3. Caspase-3-like activity has been detected in the apoptosis induced by various chemotherapeutic drugs [
36]. Caspase-3 deficiency in MCF-7 is due to a deletion mutation in exon 3 of the gene [
32,
33]. Previous studies on MCF-7 with exogenously-expressed caspase-3 indicates that caspase-3 plays an important role in apoptotic pathways [
25,
32,
33,
37]. Studies using etoposide and doxorubicin, active chemotherapeutic agents and key adjuvant drugs for breast cancer treatment, concluded that MCF-7 cells were sensitized to apoptosis only when these cells were reconstituted with caspase-3 [
38]. Chemoresistance is often caused by aberrant apoptosis that in some instances has been related to defects in caspase activation [
39,
40]. Given the importance of caspase-3 in apoptotic execution, it is then postulated that caspase-3 deficiency might significantly contribute to chemotherapeutic resistance.
In our studies, we observed manifestations of apoptosis in SPD-treated MCF-7 cells. Previous reports by Hishikawa and colleagues [
41] and Heerdt
et al., [
42] have also demonstrated similar apoptotic hallmarks in MCF-7 cells when induced with connective tissue growth factor (CTGF) and tributyrin, respectively. These suggest that the mechanism for induction of apoptosis is present and functional in MCF-7 cells, but is dependent on the external stimuli. Caspase-7 is highly related to caspase-3 and shows the same synthetic substrate specificity
in vitro [
43] suggesting that caspase-3 and -7 have possibly overlapping roles in apoptosis [
44]. Without caspase-3, SPD-treated MCF-7 cells may utilize an alternate caspase pathway to affect cell death [
45]. Here, we demonstrated that caspase-7 was activated in SPD-induced apoptosis. In MCF-7 cells treated with SPD, the 35-kDa proenzyme was cleaved into its active 17-kDa subunit. Synthesized as inactive precursors, caspases must be proteolytically cleaved to become active enzymes [
16]. Overexpression of full-length caspase-7 in the MCF-7 does not induce apoptosis, whereas the activated 17-kDa subunit induces apoptotic cell death [
46].
Activation of executioner caspases, caspase-3 or -7 results in the cleavage of critical cellular substrates and homeostatic enzymes, bringing about the manifestations of apoptosis [
16,
23]. Experimental inhibition of apoptosis by peptide caspase inhibitors presents the opportunity to investigate the importance of this protease family. When MCF-7 cells were preincubated with the caspase-7 inhibitor Ac-DEVD-CHO before treatment with SPD, apoptosis levels decreased to a level similar to controls. Cell death was thus inhibited and treated cells had morphology similar to controls, further supporting the involvement of caspase-7 in SPD-induced apoptosis.
Previous reports have found that MCF-7 cells are relatively insensitive to many chemotherapeutic agents due to the absence of caspase-3 [
38]. Our studies here have shown that the mechanism for apoptosis is functional in MCF-7 and SPD is able to induce an alternate caspase pathway, possibly via caspase-7. Tumours accumulate mutations that increase their resistance to apoptotic inducers;
e.g. abrogation of caspase-3 has been associated with acquired multidrug resistance [
47]. Therefore, finding new therapeutic agents that induce tumour cell apoptosis in a manner independent of caspase-3 may have important clinical implications. By not requiring caspase-3, SPD may evoke an apoptotic pathway different from clinical oncology drugs such as doxorubucin and etoposide [
38], thus making it a promising agent for combination chemotherapy that merits further study.