Apoptosis Protease Activator Protein-1 Expression Is Dispensable for Response of Human Melanoma Cells to Distinct Proapoptotic Agents

Abstract

Loss of expression of the apoptosis protease activator protein-1 (APAF-1) in human melanoma is thought to promote resistance to programmed cell death by preventing caspase-9 activation. However, the role of the APAF-1-dependent pathway in apoptosis activated by cellular stress and/or DNA damage has been recently questioned. We investigated APAF-1 expression in a large panel of human melanomas and assessed cellular response to several proapoptotic agents in tumors expressing or lacking APAF-1 protein. In two melanomas with wild-type p53 but with differential expression of APAF-1, treatment with camptothecin, celecoxib, or an nitric oxide synthase inhibitor (1400W) significantly modulated expression of 36 of 96 genes in an apoptosis-specific cDNA macroarray, but APAF-1 mRNA levels were not induced (in APAF-1 ؊ cells) nor upregulated (in APAF-1 ؉ cells), a finding confirmed at the protein level. Treatment with cisplatin, camptothecin, etoposide, betulinic acid, celecoxib, 1400W, and staurosporine promoted enzymatic activity not only of caspases -2, -8, and -3 but also of caspase-9 in both APAF-1 ϩ and APAF-1 ؊ tumor cells. Moreover, drug-induced caspase-9 enzymatic activity could be not only partially but significantly reduced by caspase-2, -3, and -8 -specific inhibitors in both APAF-1 ϩ and APAF-1 ؊ tumor cells. In response to 1 to 100 mol/L of cisplatin, camptothecin, or celecoxib, APAF-1 ؉ melanomas (n ‫؍‬ 12) did not show significantly increased levels of apoptosis compared with APAF-1 ؊ tumors (n ‫؍‬ 7), with the exception of enhanced apoptosis in response to a very high dose (100 mol/L) of etoposide. These results suggest that the response of human melanoma cells to different proapoptotic agents may be independent of their APAF-1 phenotype.

Apaf-1 expression in malignant melanoma

“…4,5 Moreover, our experiments correlating Apaf-1 expression with chemosensitivity were conducted blindlyone laboratory (YL) measured Apaf-1 expression and the other (SWL) assayed apoptosis and the results were compared later. Finally, we confirmed the immunoblotting results by Northern blotting and in situ hybridization 1 and, more recently, other investigators corroborated our observations using three different antibodies, [6][7][8][9] and by LOH analyses and quantitative RT-PCR. 10,11 Given that technical issues are unlikely to account for the differences between data sets, it is possible that the studies by Peltenburg and Allen did not examine enough samples to make reliable conclusions.…”

“…11 Baldi et al, Dai et al, Zannon et al and Mustika et al report a heterogeneous expression of Apaf-1, highly positive in benign nevi but weak or undetectable in 35-50% in situ melanomas and lymph node and visceral metastases. [6][7][8][9] In addition, Qin et al have also independently reported low Apaf-1 expression in metastatic melanoma cell lines used in our initial study. 12 These results, along with recent reports of low Apaf-1 expression in other melanoma cell lines, 13 are consistent with our initial findings.…”

“…Intriguingly, these results of Peltenburg et al are in contrast to a recent report showing a delayed or increased resistance of low Apaf-1-expressing melanoma cells to high doses of etoposide (as well as doxorubicin or 5FU). 8 In this context, it should be noted that the Peltenburg group has already reported mechanistic variability in the response of melanoma cells to etoposide. 22 In summary, while it is clear that Apaf-1 loss does not universally promote cell survival and is not the sole determinant of melanoma chemoresistance, 23 we think it premature to minimize its role in melanoma without further functional studies.…”

Dual inhibition of topoisomerases enhances apoptosis in melanoma cells

“…In addition, the same combination of drugs was also shown to induce apoptosis despite the fact that its extent was significantly lower in mutant p53 cells. CPTs and etoposide have been shown to induce p53-dependent mitochondrial apoptosis, with strong involvement of caspases [19,23,24]. Still, p53-independent death has also been associated with topoisomerase inhibitors [25] and it appears that several other mechanisms are involved in both processes as individual agents might interfere with other stress signaling pathways and/or directly with some organelles such as mitochondria as has been demonstrated in human gastric cancer cells [26] and cervical cancer cells [27].…”

Big wheel keeps on turning: apoptosome regulation and its role in chemoresistance

“…82 Subsequent studies performed in other laboratories have provided further support for this mechanism of Apaf-1 inactivation in metastatic melanomas, 83 although it should be noted that loss of Apaf-1 may not be the sole determinant of chemoresistance in this type of cancer. 84 Nevertheless, the fact that a substantial proportion of leukemia-derived cell lines and primary leukemic cells, 85 as well as cells from glioblastomas 86 and cervical carcinomas 87 may also inactivate Apaf-1 through a similar mechanism, further suggests that apoptosome deregulation may represent a significant event in human cancer. Indeed, loss of Apaf-1 expression is associated with tumor progression and adverse prognosis in colorectal cancer, 88 and silencing of Apaf-1 in B-cell chronic lymphocytic leukemia (B-CLL) correlates with poor prognosis in cases with concomitant p53 mutations.…”