Predominant Suppression of Apoptosome by Inhibitor of Apoptosis Protein in Non-Small Cell Lung Cancer H 460 Cells : Therapeutic Effect of a Novel Polyarginine-conjugated Smac Peptide

In the February 15, 2003, issue of Cancer Research, Yang et al. have shown that, in NSCLC-H460 cells, the apoptosome is inhibited because of the overexpression of endogenous XIAP, which was not observed in two other NSCLC cell lines, H23 and H226 (1). XIAP was found to bind and inactivate caspase-9, and the addition of an NH2-terminal Smac peptide resulted in the release of XIAP and restored caspase activation. It is proposed that XIAPdependent inhibition of caspase-9 contributes to chemotherapy resistance in H460 cells. This conclusion was based mainly on the following experiments. In cell-free assays using cytoplasmic extracts derived form these three NSCLC cell lines, the addition of dATP and cytochrome c led to the cleavage of procaspase-9, which was, however, completely sequestered and inactivated by XIAPbinding in H460 extracts, whereas this was hardly, or only partially, the case in H23 and H266 extracts. It was subsequently shown that a synthesized NH2-terminal Smac peptide fused to a membrane-permeable arginine repeat, named SmacN7(R8), can release XIAP from caspase-9 in the cell-free system. Furthermore, the sensitizing effect of SmacN7(R8) for cisplatin, in both cultured H460 cells and H460-derived tumors grown in nude mice, was presented as evidence that XIAP is responsible for caspase-9 inhibition and cisplatin resistance in H460 cells. Although the article convincingly demonstrates the potentiating effect of the Smac/DIABLO peptide on caspase activation leading to enhanced chemosensitivity, we do not agree with the conclusions drawn from this study regarding the mechanism underlying caspase-9 inhibition and the role of XIAP in NSCLC cells, because they are different from the findings that we published prior to this article (2) and that were left unmentioned. Apart from agreeing on the apparent obstruction of caspase-9 activation in H460 cells, which we also reported earlier (2), there are several discrepancies with our findings that led us to a different explanation of the mechanism underlying the inhibition of caspase-9 in H460 cells and the role of XIAP as a determinant for the response to chemotherapy in NSCLC patients. In brief, by employing various synthetic and genetic inhibitors of distinct caspases, we reported that DNA-damaging agents, such as cisplatin, fail to trigger the cleavage and activation of procaspase-9, thus placing the defect in H460 cells upstream from the processing of the proform (2). Instead, we provided evidence for the notion that caspase-8 may compensate for caspase-9 inhibition by playing an initiating role in triggering the caspase cascade in a mitochondria-dependent manner. In more detail, the following differences can be identified between the study of Yang et al. and our own findings. First, the cisplatin sensitivity of the NSCLC cells that were used differs to some extent. In our hands, the IC80 values of H460, H322, and SW1573 range between 7 and 10 M, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, concomitant with the ready induction of apoptosis that we assessed at 48–72 h post-treatment. Under these conditions, H322 cells displayed 50% lower levels of apoptosis when compared with H460 and SW1573 cells. In the experiments by Yang et al. (1), a considerable higher concentration of 10 g/ml ( 33 M) cisplatin was used that failed to trigger effective apoptosis in H460 at 24 h. The fact that the same treatment in H23 and H226 cells resulted in 20–40% apoptosis was taken as evidence for the relative resistance of H460 cells. Regardless of the discrepancies in sensitivity profiles, we believe this conclusion is not supported by the facts presented, because only three cell lines were studied and also in light of our findings that demonstrated no substantial differences in cisplatin sensitivity in NSCLC H460, SW1573 and H322 cells. Moreover, we provided evidence for caspase-9 inhibition in H460, H322, and SW1573, suggesting that this may be a more general mechanism of drug resistance in NSCLC cell lines (2). Second, in the cell-free assays used by Yang et al. (1), cytochrome c and ATP were directly used to activate the apoptosome; this is a rather artificial system when compared with the response in intact cells in which DNA-damaging agents subsequently trigger signaling cascades that disrupt mitochondria, leading to the release of cytochrome c and Smac/DIABLO (3, 4) and to the formation of the active apoptosome. Questions that remain unaddressed in the cell-free assays are whether endogenous Smac/ DIABLO and cytochrome c are released in H460 cells, and whether or not this is sufficient for the release of IAPs (i.e., XIAP) from active caspase-9. In addition, the authors used the activation of caspase-3, determined by cleavage of fluorescent DEVD-MCA, as an end point in studying the effect of the SmacN7 peptide on the release of XIAP from active caspase-9, whereas evidence for the restored activation of caspase-9 by cleavage of fluorescent-caspase-9 substrate (LEHD) is missing in the cell-free assays as well as in extracts obtained from H460 cells treated with cisplatin. Furthermore, the lack of analysis on a possible upstream role of other caspases in relation to caspase-3 activation precludes any definitive conclusion and does not address our finding that cisplatin exposure leads to an increase in DEVDase activity that can be abrogated by blocking caspase-8 activation. Last but not least, we have recently reported that the expression of c-IAP1, c-IAP2, and XIAP in tumor samples derived from 55 patients with advanced NSCLC did not correlate with the response to cisplatinbased chemotherapy (5), indicating the existence of differences between preclinical and clinical studies, and pointing to the involvement of other or additional genetic factors in determining treatment outcome. In summary, the neutralization of proteins belonging to the family of IAPs known to block the activity of multiple caspases is expected to result in lowering the threshold for caspase activation induced by chemotherapy. In this context, chemosensitization on treatment with the SmacN7 peptide can be readily explained and is a finding that we support. In fact, in a panel of NSCLC cells that we have analyzed for IAP expression, a number of IAPs appear to be overexpressed that may be related to chemoresistance in NSCLC cells in vitro, although their possible predictive value in the clinic remains to be further investigated. However, as explained above, we do not agree with the model proposed by Yang et al. that the XIAP-dependent blockade of cleaved caspase-9 is the major determinant of chemoresistance in H460 cells but postulate that one or more factors upstream of caspase-9 cleavage, which may be involved in the formation of the apoptosome, is the cause of caspase-9 inhibition. Whether such (a) yet Received 4/28/03; revised 6/12/03; accepted 6/12/03. 1 The abbreviations used are: NSCLC, non-small cell lung cancer; XIAP, X-linked IAP; IAP, inhibitor of apoptosis protein. 2 Unpublished results. 6566