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Anti-apoptotic Bcl-2 family proteins, in particular, Mcl-1, are known to play a critical role in resistance of human melanoma cells to induction of apoptosis by endoplasmic reticulum stress and other agents. The present study examined whether the BH3 mimetics, Obatoclax and ABT-737, which inhibit multiple anti-apoptotic Bcl-2 family proteins, would overcome resistance to apoptosis. We report that both agents induced a strong unfolded protein response (UPR) and that RNAi knockdown of UPR signalling proteins ATF6, IRE1α and XBP-1 inhibited Mcl-1 upregulation and increased sensitivity to the agents. These results demonstrate that inhibition of anti-apoptotic Bcl-2 proteins by Obatoclax and ABT-737 appears to elicit a protective feedback response in melanoma cells, by upregulation of Mcl-1 via induction of the UPR. We also report that Obatoclax, but not ABT-737, strongly induces autophagy, which appears to play a role in determining melanoma sensitivity to the agents.

Although the introduction of selective v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) inhibitors has been a major advance in treatment of metastatic melanoma, approximately 50% of patients have limited responses including stabilization of disease or no response at all. This study aims to identify a novel means of overcoming resistance of melanoma to killing by BRAF inhibitors. We examined the influence of the BH3-mimetic ABT-737 on induction of apoptosis by the selective BRAF inhibitor PLX4720 in melanoma cells with or without BRAF V600E mutation. Included were cell lines established from four patients before and during treatment with selective BRAF inhibitors and 3D spheroids derived from these cell lines. Cell lines with no or low sensitivity to PLX4720 underwent synergistic increases and increased rates of apoptosis when combined with ABT-737. This degree of synergism was not seen in cell lines without BRAF V600E mutations. Apoptosis was mediated through the mitochondrial pathway and was due in part to upregulation of Bim as shown by inhibition of apoptosis following small interfering RNA knockdown of Bim. Similar effects were seen in cell lines established from patients prior to treatment but not in lines from patients clinically resistant to the selective BRAF inhibitors and in 3D spheroids derived from these cell lines. These results suggest that combination of selective BRAF inhibitors with ABT-737 or the related orally available compound ABT-263 may increase the degree and rate of responses in previously untreated patients with V600E melanoma but not in those with acquired resistance to these agents.

HDACs are viewed as enzymes used by cancer cells to inhibit tumor suppressor mechanisms. In particular, we discuss their role as suppressors of apoptosis in melanoma cells and as mediators of resistance to selective BRAF inhibitors. Synergistic increases in apoptosis are seen when pan-HDAC inhibitors are combined with selective BRAF inhibitors. Moreover, cell lines from patients with acquired resistance to Vemurafenib undergo PLX4720 induced apoptosis when combined with pan-HDAC inhibitors. The mechanisms of upregulation of HDACs and the mechanisms involved in HDACi reversal of resistance to apoptosis are as yet poorly understood., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Metastatic melanoma represents a major clinical problem. Its incidence continues to rise in western countries and there are currently no curative treatments. While mutation of the P53 tumour suppressor gene is a common feature of many types of cancer, mutational inactivation of P53 in melanoma is uncommon; however, its function often appears abnormal., Methods: In this study whole genome bead arrays were used to examine the transcript expression of P53 target genes in extracts from 82 melanoma metastases and 6 melanoma cell lines, to provide a global assessment of aberrant P53 function. The expression of these genes was also examined in extracts derived from diploid human melanocytes and fibroblasts., Results: The results indicated that P53 target transcripts involved in apoptosis were under-expressed in melanoma metastases and melanoma cell lines, while those involved in the cell cycle were over-expressed in melanoma cell lines. There was little difference in the transcript expression of P53 target genes between cell lines with null/mutant P53 compared to those with wild-type P53, suggesting that altered expression in melanoma was not related to P53 status. Similarly, down-regulation of P53 by short-hairpin RNA (shRNA) had limited effect on P53 target gene expression in melanoma cells, whereas there were a large number of P53 target genes whose mRNA expression was significantly altered by P53 inhibition in melanocytes. Analysis of whole genome gene expression profiles indicated that the ability of P53 to regulate genes involved in the cell cycle was significantly reduced in melanoma cells. Moreover, inhibition of P53 in melanocytes induced changes in gene expression profiles that were characteristic of melanoma cells and resulted in increased proliferation. Conversely, knockdown of P53 in melanoma cells resulted in decreased proliferation., Conclusions: These results indicate that P53 target genes involved in apoptosis and cell cycle regulation are aberrantly expressed in melanoma and that this aberrant functional activity of P53 may contribute to the proliferation of melanoma.

Purpose: To examine mechanisms that determine long-term responses of B-RAF(V600E) melanoma cells to B-RAF inhibitors., Experimental Design: B-RAF(V600E) melanoma cells were exposed to the B-RAF inhibitor PLX4720 for prolonged periods to select for cells resistant to apoptosis induced by the inhibitor. The resultant cells were analyzed for activation of extracellular signal regulated kinase (ERK), MAP/ERK kinase (MEK), and Akt, and related signals. Their roles in survival of the cells were also examined., Results: B-RAF(V600E) melanoma cells selected for resistant to PLX4720-induced apoptosis retained the V600E mutation in B-RAF, and proliferated steadily in the presence of the inhibitor, albeit with slow growth rate. These cells displayed high levels of ERK activation, that is, at least in part, independent of the conventional RAF/MEK/ERK pathway, as MEK activation was low and inhibition of MEK did not significantly block activation of ERK. In contrast, extracellular signals appeared involved. This was associated with elevated activation of the phosphoinositide 3-kinase (PI3k)/Akt pathway and could be inhibited by serum starvation and inhibition of PI3k/Akt. Inhibition of MEK did not impact on survival of these cells, whereas serum starvation, inhibition of PI3K/Akt, and inhibition of ERK1/2 reduced their viability., Conclusions: These results indicate that sensitivity to induction of apoptosis may be a major determinant of long-term responses of B-RAF(V600E) melanomas to specific inhibitors and suggest that rebound melanoma growth after initial treatment with the inhibitors may not be responsive to MEK inhibitors, but may be susceptible to inhibition of the PI3k/Akt pathway., (©2010 AACR.)

Wild-type p53 is commonly expressed in melanoma but does not appear to be effective in the induction of apoptosis. One explanation is that p53 is targeted for degradation by the E3 ligase MDM2. However, we found in this study that blockade of the interaction of p53 and MDM2 by the MDM2 antagonist nutlin-3 in melanoma cells did not induce apoptosis, even though it upregulated p53 and its proapoptotic targets. Nevertheless, nutlin-3 enhanced TRAIL-induced apoptosis as a result of p53-mediated upregulation of TRAIL-R2. Unexpectedly, nutlin-3 upregulated Mcl-1, which attenuated apoptotic signaling triggered by TRAIL, and inhibited apoptosis induced by the microtubule-targeting drug docetaxel. The increase in Mcl-1 was related to a p53-independent transcriptional mechanism, but stabilization of the Mcl-1 protein played a dominant role, as nutlin-3 upregulated the Mcl-1 protein to a much greater extent than the Mcl-1 mRNA, and this was associated with prolonged half-life time and reduced ubiquitination of the protein. Knockdown of p53 blocked the upregulation of the Mcl-1 protein, indicating that p53 plays a critical role in the stabilization of Mcl-1. The contrasting effects of nutlin-3 on TRAIL- and docetaxel-induced apoptosis were confirmed in fresh melanoma isolates. Collectively, these results show that nutlin-3 may be a useful agent in combination with TRAIL and, importantly, uncover a novel regulatory effect of p53 on the expression of Mcl-1 in melanoma cells on treatment with nutlin-3, which may antagonize the therapeutic efficacy of other chemotherapeutic drugs in addition to docetaxel in melanoma., (©2010 AACR.)

Although it is conventionally regarded as an inflammatory caspase, recent studies have shown that caspase-4 plays a role in induction of apoptosis by endoplasmic reticulum (ER) stress. We report here that activation of caspase-4 is also involved in induction of apoptosis by TNF-related apoptosis-inducing ligand (TRAIL) in human melanoma cells. Treatment with TRAIL resulted in activation of caspase-4. This appeared to be mediated by caspase-3, in that caspase-4 was activated later than caspase-8, -9, and -3, and that inhibition of caspase-3 blocked TRAIL-induced caspase-4 activation. Notably, TRAIL triggered ER stress in melanoma cells as shown by up-regulation of the GRP78 protein and the spliced form of XBP-1 mRNA. This seemed to be necessary for activation of caspase-4, as activation of caspase-3 by agents that did not trigger ER stress did not cause activation of caspase-4. Importantly, inhibition of caspase-4 also partially blocked caspase-3 activation, suggesting that activation of caspase-4 may be positive feed-back mechanism to further enhance caspase-3 activation. Collectively, these results show that activation of caspase-4 contributes to TRAIL-induced apoptosis and is associated with induction of ER stress by TRAIL in melanoma cells, and may have important implications for improving therapeutic efficacies of TRAIL in melanoma.

Bim is known to be critical in killing of melanoma cells by inhibition of the RAF/MEK/ERK pathway. However, the potential role of the most potent apoptosis-inducing isoform of Bim, Bim(S), remains largely unappreciated. Here, we show that inhibition of the mutant B-RAF(V600E) triggers preferential splicing to produce Bim(S), which is particularly important in induction of apoptosis in B-RAF(V600E) melanoma cells. Although the specific B-RAF(V600E) inhibitor PLX4720 upregulates all three major isoforms of Bim, Bim(EL), Bim(L), and Bim(S), at the protein and mRNA levels in B-RAF(V600E) melanoma cells, the increase in the ratios of Bim(S) mRNA to Bim(EL) and Bim(L) mRNA indicates that it favours Bim(S) splicing. Consistently, enforced expression of B-RAF(V600E) in wild-type B-RAF melanoma cells and melanocytes inhibits Bim(S) expression. The splicing factor SRp55 appears necessary for the increase in Bim(S) splicing, as SRp55 is upregulated, and its inhibition by small interfering RNA blocks induction of Bim(S) and apoptosis induced by PLX4720. The PLX4720-induced, SRp55-mediated increase in Bim(S) splicing is also mirrored in freshly isolated B-RAF(V600E) melanoma cells. These results identify a key mechanism for induction of apoptosis by PLX4720, and are instructive for sensitizing melanoma cells to B-RAF(V600E) inhibitors.

Past studies have identified a number of distinct mechanisms that contribute to the resistance of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). In this report we show that cystatin B is another endogenous inhibitor of TRAIL-induced apoptosis. Cystatin B-deficient melanoma cell lines established by shRNA knockdown displayed increased apoptosis that was associated with enhanced activation of caspase-8 induced by TRAIL. This was not related to the inhibitory effect of cystatin B on the lysosomal cysteine proteases, cathepsin B and L, as they did not have a role in TRAIL-induced apoptosis in most melanoma cell lines even when cystatin B was inhibited. Instead, sensitization of melanoma cells to TRAIL-induced apoptosis by inhibition of cystatin B appeared associated with decreased stability of FLIP(L) as the levels of FLIP(L) were reduced because of shortened half-life time in melanoma cells deficient in cystatin B. In contrast, over-expression of cystatin B increased the levels of FLIP(L), decreased the amount of the E3 ligase Itch associated with FLIP(L), and reduced FLIP(L) ubiquitination. Inhibition of Itch by siRNA restored the levels of FLIP(L) and blocked sensitization to TRAIL-induced apoptosis associated with deficiency in cystatin B. Taken together, these results indicate that cystatin B regulates Itch-mediated degradation of FLIP(L) and thereby TRAIL-induced apoptosis in melanoma cells.

Background: Past studies have shown that sensitivity of melanoma cells to TRAIL-induced apoptosis is largely correlated with the expression levels of TRAIL death receptors on the cell surface. However, fresh melanoma isolates and melanoma tissue sections express generally low levels of death receptors for TRAIL. The clinical potential of TRAIL in the treatment of melanoma may therefore be limited unless given with agents that increase the cell surface expression of TRAIL death receptors. 2-Deoxy-D-glucose (2-DG) is a synthetic glucose analogue that inhibits glycolysis and glycosylation and blocks cell growth. It has been in clinical evaluation for its potential use as an anticancer agent. In this study, we have examined whether 2-DG and TRAIL interact to enhance their cytotoxicity towards melanoma cells., Results: 2-DG did not kill melanoma cells, but enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. This was associated with increased activation of the caspase cascade and mitochondrial apoptotic pathway, and was blocked by inhibition of TRAIL-R2, and to a lesser extent, inhibition of TRAIL-R1. Treatment with 2-DG up-regulated TRAIL death receptors, in particular, TRAIL-R2, on the melanoma cell surface. Up-regulation of TRAIL-R2 was due to increased transcription that was not dependent on the transcription factors, p53 and CHOP. Instead, the IRE1 alpha and ATF6 pathways of the unfolded protein response that were activated by 2-DG appeared to be involved. Moreover, XBP-1, which is known to be transcriptionally regulated by ATF6 and functionally activated by IRE1 alpha, was found to play an important role in 2-DG-mediated transcriptional up-regulation of TRAIL-R2 in melanoma cells., Conclusion: These results indicate that 2-DG sensitizes human melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-2 via the ATF6/IRE1 alpha/XBP-1 axis of the unfolded protein response. They suggest that 2-DG is a promising agent to increase the therapeutic response to TRAIL in melanoma.

Immunotherapy based on T cell responses to the tumor is believed to involve killing of cancer cells by induction of apoptosis. The predominant mechanisms are death ligand-induced signaling mainly by TNF-related apoptosis-inducing ligand (TRAIL) mediated by CD4 T cells, monocytes and dendritic cells, and perforin/granzyme mediated apoptosis mediated by CD8 T cells and NK cells. Resistance against TRAIL involves loss of TRAIL death receptors and/or activation of the MEK and/or Akt signal pathways. Resistance to CD8 CTL responses also involves activation of the MEK and/or Akt pathways. Apoptosis induced by immune responses is regulated by the Bcl-2 family of proteins. Many reagents have been developed against the Bcl-2 antiapoptotic proteins and clinical trials combining them with immunotherapy are awaited. The second group of agents that regulate the Bcl-2 family of proteins are the signal pathway inhibitors. Clinical trials with inhibitors of RAS, RAF or MEK are in progress and would appear an exciting combination with immunotherapy. One of the main drivers of resistance to apoptosis are adaptive mechanisms that allow cancer cells to overcome endoplasmic reticulum (ER) stress. These adaptive mechanisms inhibit practically all known apoptotic pathways and create an acidic environment that may reduce infiltration of lymphocytes against the tumor. The signal pathway inhibitors may be effective against these adaptive processes but additional agents that target ER stress pathways are in development. In conclusion, combination of immunotherapy with agents that target antiapoptotic mechanisms in cancer cells offers a new approach that requires evaluation in clinical trials.

Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER) stress, and this is associated with up-regulation of the antiapoptotic proteins Bcl-2 and Mcl-1. In this report, we show that the BH3 mimetic obatoclax potently overcomes resistance of melanoma cells to apoptosis induced by ER stress. Obatoclax, as a single agent at nanomolar concentrations, was relatively ineffective in the induction of apoptosis in melanoma cells, but treatment with obatoclax at these concentrations in combination with the ER stress inducer tunicamycin (TM) or thapsigargin markedly enhanced apoptotic cell death. This was primarily because of the inhibition of Mcl-1 by obatoclax, in that cotreatment with TM and another BH3 mimetic ABT737, which does not antagonize Mcl-1, caused only minimal increases in apoptosis. Moreover, overexpression of Mcl-1 inhibited apoptosis to greater degrees than overexpression of Bcl-2. In addition to direct inhibition of Mcl-1 by obatoclax, the combination of obatoclax and TM caused strong up-regulation of the BH3-only protein Noxa. Small RNA interference knockdown of Noxa partially inhibited apoptosis induced by cotreatment with obatoclax and TM. Similarly, knockdown of Bak also blocked induction of apoptosis by the compounds. The Mcl-1/Bak interaction seemed to be disrupted more efficiently in melanoma cells cotreated with obatoclax and TM. Taken together, these results identify obatoclax as a potent agent that overcomes resistance of melanoma cells to ER stress-induced apoptosis and seem to have important implications in the use of BH3 mimetics in the treatment of melanoma.

We studied potential interactions between the endoplasmic reticulum (ER) stress response and the MEK/ERK pathway. Induction of ER stress did not trigger significant apoptosis, but caused rapid activation of ERK1/2 in gastric cancer cells. Inhibition of MEK enhanced ER stress-induced apoptosis via a caspase-dependent, mitochondria-mediated mechanism. This was associated with blockage of ER stress-mediated up-regulation of GRP78. The latter appeared to be critical in antagonizing the apoptosis-inducing potential of ER stress. Thus, activation of MEK/ERK by ER stress is necessary for induction of GRP78 that protects against apoptosis in gastric cancer cells submitted to ER stress.

Our previous studies revealed that Docetaxel-induced apoptosis of melanoma cells is entirely dependent on activation of the JNK signalling pathway. Here, we show that Docetaxel-induced apoptosis is mediated by induction of ER stress. This was shown by Docetaxel-induced activation of proteins involved in ER stress signalling namely GRP78, ATF6, IRE1alpha, and PERK/eIF2alpha. Knockdown of IRE1alpha by siRNA markedly inhibited Docetaxel-induced JNK activation and downstream targets of JNK indicating that activation of IRE1alpha was upstream of activation of the JNK. Co-immunoprecipitation experiments showed that activation of JNK is due to activation of ASK1 through formation of an IRE1alpha-TRAF2-ASK1 complex. ER stress mediated activation of the JNK pathway is downstream of activation of PKCdelta in that downregulation of PKCdelta expression using specific PKCdelta siRNA significantly inhibited Docetaxel-induced activation of IRE1alpha and the JNK pathway. These findings provide new insights to understand the mode of action of taxanes in treatment of human melanoma.

We have previously shown that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress-induced apoptosis, and this involves activation of the mitogen-activated protein/extracellular signal-regulated kinase (MEK)/ERK signaling pathway and expression of the apoptosis repressor with caspase recruitment domain (ARC) protein in the cells. In the present study, we show that up-regulation of the antiapoptotic Bcl-2 family member Mcl-1 is another mechanism critical for protection of melanoma cells against ER stress-induced apoptosis. Inhibition of Mcl-1 by small interference RNA (siRNA) rendered melanoma cells sensitive to apoptosis induced by the ER stress inducers thapsigargin and tunicamycin, but this sensitization was partially reversed by siRNA knockdown of PUMA or Noxa, as shown in Mcl-1-deficient melanoma cells. Both PUMA and Noxa were increased by ER stress through transcriptional up-regulation, but only up-regulation of Noxa was dependent on p53, whereas up-regulation of PUMA seemed to be mediated by a p53-independent mechanism(s). Up-regulation of Mcl-1 was also due to increased transcription that involved the IRE1alpha and activating transcription factor 6 signaling pathways of the unfolded protein response. In addition, activation of the MEK/ERK signaling pathway seemed to be necessary for optimal up-regulation of Mcl-1. Taken together, these results reveal the mechanisms of resistance of melanoma cells to apoptosis induction mediated by BH3-only proteins upon ER stress, and identify Mcl-1 as a target for the treatment of melanoma in combination with therapeutics that induce ER stress.

The molecular changes involved in the induction of apoptosis by vincristine in melanoma have not yet been well defined. Two human melanoma cell lines showing moderate (Mel-RM) and high (IgR3) sensitivity to vincristine were selected from a panel of eight melanoma lines for analysis. Induction of apoptosis was caspase dependent, and was associated with increases in mitochondrial membrane permeability. Vincristine upregulated the expression of Bax, Bak, PUMA, Noxa, p53 and p21 proteins, and downregulated and/or phosphorylated the Bcl-2 protein. Inhibitors of the Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein kinase, significantly inhibited vincristine-induced apoptosis in both IgR3 and Mel-RM cells. In addition, vincristine induced phosphorylation and reduction in Bcl-2 was prevented by an inhibitor of JNK. Downregulation of mRNA for p53, PUMA or Bim by RNA interference had little or no influence on vincristine-induced apoptosis in IgR3 cells. In addition, silencing Bim mRNA did not affect vincristine-induced apoptosis in Mel-RM cells. These results suggest that vincristine-induced apoptosis of at least some melanoma cell lines is dependent on the activation of JNK. The results are consistent with the phosphorylation of Bcl-2 protein, resulting in the activation of Bax/Bak, release of cytochrome c from the mitochondria and the resulting activation of caspases.

Although cisplatin has been shown to induce both apoptosis and necrosis in cancer cells, the potential interconnections between these modes of cell death induced by the drug remain unknown. We studied this phenomenon in gastric cancer cell lines and identified one cell line (SGC-7901) that underwent apoptosis, and another cell line (BGC-823) that primarily underwent nonapoptotic cell death, in response to cisplatin. Apoptosis in cisplatin-treated SGC-7901 cells seemed to be caspase dependent and was mediated, at least in part, by the BH3-only protein, Noxa. This was evidenced by the rapid upregulation of Noxa and inhibition of apoptosis by small interfering RNA knockdown of Noxa. Nonapoptotic cell death induced by cisplatin in BGC-823 cells was characterized by lack of DNA fragmentation, delayed externalization of phosphatidylserine, caspase independence, plasma membrane disruption, and intracellular vacuole formation, indicative of necrosis. Surprisingly, blockage of apoptosis induction by a general caspase inhibitor or by Noxa small interfering RNA in SGC-7901 failed to protect against cisplatin-induced cell death. Under such conditions, SGC-7901 cells displayed cellular features associated with necrosis. Cisplatin-induced apoptosis, thus, seems to precede necrosis when the apoptotic machinery is operative. When the apoptosis program is defective, necrotic cell death takes place as an alternative pathway leading to cell demise. Induction of different modes of cell death that are interrelated in the same cells by cisplatin has the potential to be exploited in formulating new adjuvant cancer therapies.

We have shown previously that most melanoma cell lines are insensitive to endoplasmic reticulum (ER) stress-induced apoptosis, but resistance can be reversed through activation of caspase-4 by inhibition of the MEK/ERK pathway. We report in this study that apoptosis was induced by the ER stress inducer thapsigargin or tunicamycin via a caspase-8-mediated pathway in the melanoma cell line Me1007, although the MEK/ERK pathway was activated in this cell line. The high sensitivity of Me1007 to ER stress-induced apoptosis was associated with low expression levels of the apoptosis repressor with caspase recruitment domain (ARC) protein that was expressed at relatively high levels in the resistant melanoma cell lines. Transfection of cDNA encoding ARC into Me1007 cells inhibited both caspase-8 activation and apoptosis induced by thapsigargin or tunicamycin. In contrast, inhibition of ARC by small interfering RNA knockdown sensitized the resistant melanoma cell lines to ER stress-induced apoptosis, which was inhibitable by blockage of caspase-8 activation. Both exogenous and endogenous ARC seemed to predominantly locate to the cytoplasm and mitochondria and could be coimmunoprecipitated with caspase-8. Taken together, ER stress can potentially activate multiple apoptosis signaling pathways in melanoma cells in a context-dependent manner. Whereas the MEK/ERK signaling pathway plays an important role in inhibiting ER stress-induced caspase-4 activation, ARC seems to be critical in blocking activation of caspase-8 in melanoma cells subjected to ER stress.

We have previously shown that Docetaxel-induced variable degrees of apoptosis in melanoma. In this report, we studied the beta-tubulin repertoire of melanoma cell lines and show that class III beta-tubulin expression correlated with Docetaxel-resistance. Sensitive cells showed low levels of class III beta-tubulin with little microtubular incorporation, whereas class III beta-tubulin expression was higher in resistant cells and was incorporated into the cytoskeleton. As proof of concept, abrogation of class III by siRNA reverted Docetaxel-resistant cells to a sensitive phenotype, restoring the microtubular polymerisation response and promoting high levels of apoptosis through Bax activation. These results suggest that phenotypic expression of beta-tubulin class III in melanoma may help identify patients with melanoma that can respond to taxanes.

Temozolomide (TMZ), a DNA alkylating agent used in the treatment of melanoma, is believed to mediate its effect by addition of a methyl group to the O(6) position of guanine in DNA. Resistance to the agent may be in part due to the activity of O(6)-methylguanine-DNA methyl transferase (MGMT). In the present study, we show that sensitivity of melanoma cells to TMZ was dependent on their p53 status and levels of MGMT. Analysis of the mechanisms underlying reduced viability showed no evidence for induction of apoptosis even though marked levels of apoptosis was seen in TK6 lymphoma cells. Sensitivity of melanoma cells was associated with p53-dependent G2/M cell cycle arrest and induction of senescence. To verify the role of p53, the assays were repeated in presence of pifithrin-alpha, an inhibitor of p53. This resulted in increased viability of melanoma cells with wild-type p53 and reversed G2/M cell cycle arrest. Paradoxically, apoptosis was increased in melanoma but decreased as expected in TK6 lymphoma cells. These results are consistent with the view that TMZ is relatively ineffective against melanoma due to defective apoptotic signalling resulting from activation of p53. The nature of the defects in apoptotic signalling remains to be explored.

We have previously reported that sensitivity of melanoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is largely correlated with the levels of expression of TRAIL death receptors, in particular, TRAIL-R2 on the cell surface. However, fresh melanoma isolates and melanoma tissue sections express, in general, low levels of death receptors for TRAIL. We show in this study that the endoplasmic reticulum stress inducer, thapsigargin (TG), selectively up-regulated TRAIL-R2 and enhanced TRAIL-induced apoptosis in melanoma cells. However, the TRAIL-R2 pathway did not appear to be involved in induction of apoptosis by TG alone. Up-regulation of TRAIL-R2 appeared to be cooperatively mediated by the inositol-requiring transmembrane kinase and endonuclease 1alpha (IRE1alpha)- and activation of transcription factor (ATF)-6-signaling pathways of the unfolded protein response (UPR) and the transcription factor CCAAT/enhancer-binding protein-homologous protein (CHOP). The latter played a critical role in the initial phase of the increase in TRAIL-R2 as small interfering RNA (siRNA) knockdown of CHOP blocked up-regulation of TRAIL-R2 only at a relatively early stage (16 h) after exposure to TG. In contrast, IRE1alpha and ATF6 appeared to be crucial in maintaining the increased levels of TRAIL-R2 in that siRNA knockdown of IRE1alpha or ATF6 had no effect on the increase in TRAIL-R2 at the initial phase, but blocked TRAIL-R2 up-regulation at a relatively late stage (36 h). Our results indicate that modulation of the UPR may be useful in sensitizing melanoma cells to TRAIL-induced apoptosis by up-regulation of TRAIL-R2.

Past studies have shown that activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK is a common cause for resistance of melanoma cells to death receptor-mediated or mitochondria-mediated apoptosis. We report in this study that inhibition of the MEK/ERK pathway also sensitizes melanoma cells to endoplasmic reticulum (ER) stress-induced apoptosis, and this is mediated, at least in part, by caspase-4 activation and is associated with inhibition of the ER chaperon glucose-regulated protein 78 (GRP78) expression. Treatment with the ER stress inducer tunicamycin or thapsigargin did not induce significant apoptosis in the majority of melanoma cell lines, but resistance to these agents was reversed by the MEK inhibitor U0126 or MEK1 small interfering RNA (siRNA). Induction of apoptosis by ER stress when MEK was inhibited was caspase dependent with caspase-4, caspase-9, and caspase-3 being involved. Caspase-4 seemed to be the apical caspase in that caspase-4 activation occurred before activation of caspase-9 and caspase-3 and that inhibition of caspase-4 by a specific inhibitor or siRNA blocked activation of caspase-9 and caspase-3, whereas inhibition of caspase-9 or caspase-3 did not inhibit caspase-4 activation. Moreover, overexpression of Bcl-2 inhibited activation of caspase-9 and caspase-3 but had minimal effect on caspase-4 activation. Inhibition of MEK/ERK also resulted in down-regulation of GRP78, which was physically associated with caspase-4, before and after treatment with tunicamycin or thapsigargin. In addition, siRNA knockdown of GRP78 increased ER stress-induced caspase-4 activation and apoptosis. Taken together, these results seem to have important implications for new treatment strategies in melanoma by combinations of agents that induce ER stress and inhibitors of the MEK/ERK pathway.

Our previous studies showed that docetaxel-induced apoptosis of human melanoma cells was dependent on the activation of the c-jun NH(2)-terminal kinase (JNK) signaling pathway but was inhibited by the extracellular signal-regulated kinase (ERK)-1/2 pathway. However, the mechanisms by which these pathways were modulated by docetaxel were not clear. We report here that docetaxel induces activation of protein kinase C (PKC) signaling differentially through PKCepsilon and PKCdelta isoforms. Activation of PKCepsilon was most marked in docetaxel-resistant cells and paralleled the activation of the ERK1/2 pathway. Inhibition of PKCepsilon by small interfering RNA molecules resulted in down-regulation of phosphorylated ERK1/2 and sensitization of cells to docetaxel-induced apoptosis. Experiments also showed that beta-tubulin class III, a molecular target of docetaxel, coimmunoprecipitated with PKCepsilon and colocalized in confocal microscopic studies. In contrast to PKCepsilon, high levels of activated PKCdelta were associated with activation of the JNK pathway and sensitivity to docetaxel. Activation of PKCdelta seemed to be upstream of JNK because inhibition of PKCdelta by small interfering RNA abrogated activation of the JNK pathway. Although PKCdelta could be activated in resistant cells, downstream activation of JNK and c-Jun did not occur. In summary, these results suggest that the outcome of docetaxel-induced apoptotic events in human melanoma cells depends on their PKC isoform content and signaling responses. PKCepsilon was associated with prosurvival signaling through ERK, whereas PKCdelta was associated with proapoptotic responses through JNK activation.

Purpose: Given that inhibitors of mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK) are being introduced into treatment for melanoma, the present study was carried out to better understand the mechanism by which they may induce apoptosis of melanoma cells., Experimental Design: A panel of human melanoma cell lines and fresh melanoma isolates was assessed for their sensitivity to apoptosis induced by the MEK inhibitor U0126. The apoptotic pathways and regulatory mechanisms involved were examined by use of the inhibitor and small interfering RNA (siRNA) techniques., Results: Inhibition of MEK induced apoptosis in the majority of melanoma cell lines through a mitochondrial pathway that was associated with the activation of Bax and Bak, release of mitochondrial apoptogenic proteins, and activation of caspase-3. However, apoptosis was independent of caspases and instead was associated with mitochondrial release of AIF as shown by the inhibition of apoptosis when AIF was knocked down by siRNA. Inhibition of MEK resulted in the up-regulation of the BH3-only proteins PUMA and Bim and down-regulation of the antiapoptotic protein Mcl-1. These changes were critical for the induction of apoptosis by U0126 as siRNA knockdown of PUMA or Bim inhibited apoptosis, whereas siRNA knockdown of Mcl-1 increased apoptosis particularly in the apoptosis-resistant cell lines., Conclusions: Apoptosis of melanoma cells induced by the inhibition of the MEK/ERK pathway is mediated by the up-regulation/activation of PUMA and Bim and down-regulation of Mcl-1. Release of AIF rather than the activation of caspases seems to be the mediator of apoptosis. Our results suggest that cotargeting Mcl-1 and the MEK/ERK pathway may further improve treatment results in melanoma.

We have reported previously low expression of death receptors for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in fresh isolates and tissue sections of melanoma. This seemed to correlate with relative resistance of freshly isolated melanoma cells to TRAIL-induced apoptosis. We show in this study that the endoplasmic reticulum (ER) stress inducer, tunicamycin, selectively up-regulated the cell surface expression of TRAIL-R2, but not other members of the TNF receptor family, and enhanced TRAIL-induced apoptosis in cultured melanoma cells and fresh melanoma isolates. Tunicamycin-mediated sensitization of melanoma cells to TRAIL-induced apoptosis was associated with increased activation of the caspase cascade and reduction in mitochondrial membrane potential and was inhibited by a recombinant TRAIL-R2/Fc chimeric protein. Up-regulation of TRAIL-R2 on the melanoma cell surface was associated with increased transcription of TRAIL-R2 and its total protein levels. Two signaling pathways of the ER stress-induced unfolded protein response mediated by inositol-requiring transmembrane kinase and endonuclease 1alpha (IRE1alpha) and activation of transcription factor 6 (ATF6), respectively, seemed to be involved. In one melanoma line, there was clear evidence of activation of the IRE1alpha pathway, and small interfering RNA (siRNA) knockdown of IRE1alpha substantially reduced the up-regulation of TRAIL-R2. Similarly, there was evidence for the activation of the ATF6 pathway, and siRNA knockdown of ATF6 had a delayed effect on TRAIL-R2 expression in one but not another melanoma cell line. Moreover, the transcription factor CCAAT/enhancer-binding protein homologous protein seemed to be involved in the up-regulation of TRAIL-R2 by tunicamycin, but its role varied between different melanoma lines. Taken together, our results suggest that agents that induce ER stress may enhance TRAIL-R2 expression and increase the therapeutic response to TRAIL in melanoma.

Purpose: Our studies have shown variable sensitivity of cultured melanoma cells to docetaxel. To better understand this response, we studied the role of signal transduction pathways in modulating docetaxel-induced melanoma killing., Experimental Design: Involvement of c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase, and Akt signaling was studied by evaluating their extent of activation in melanoma cells after treatment with docetaxel. The effect of their activation on docetaxel-induced apoptosis was assessed using biochemical inhibitors of the pathways and Western blot analysis of proteins involved., Results: Docetaxel induced activation of both JNK and ERK1/2 but not p38 mitogen-activated protein kinase or Akt kinases. Apoptosis was dependent on activation of JNK and mediated through activation of caspase-2 and caspase-dependent changes in Bax and Bak. The levels of activated JNK in individual lines showed a close correlation with the levels of apoptosis. In contrast, activation of ERK1/2 by docetaxel inhibited apoptosis and the levels of activation in individual lines were inversely correlated to the degree of apoptosis. Studies on the Bcl-2 family proteins seemed to reflect changes induced by activation of JNK and ERK1/2 pathways. Docetaxel-induced JNK activation was required for Bcl-2 phosphorylation as well as caspase-2-dependent activation of Bax and Bak and subsequent mitochondrial release of apoptosis-inducing factor and cytochrome c. In contrast, activation of ERK1/2 resulted in degradation of BH3-only protein Bim and phosphorylation of Bad., Conclusions: These studies provide further insights into sensitivity of melanoma cells to taxanes and provide a basis for the current rationale of combining taxanes with inhibitors of the Raf-ERK1/2 pathway.

Taxanes have a broad spectrum of activity against various human cancers, including melanoma. In this study, we have examined the molecular mechanism of docetaxel-induced apoptosis of human melanoma. We report that docetaxel induced varying degrees of apoptosis in a panel of melanoma cell lines but not in normal fibroblasts. Induction of apoptosis was caspase dependent and associated with changes in mitochondrial membrane potential that could be inhibited by overexpression of Bcl-2. Docetaxel induced changes in Bax that correlated with sensitivity to docetaxel-induced apoptosis. These changes in Bax were not inhibited by overexpression of Bcl-2. Kinetic studies of caspase-2 activation by Western blotting and fluorogenic assays revealed that activation of caspase-2 seemed to be the initiating event. Inhibition of caspase-2 with z-VDVAD-fmk or by small interfering RNA knockdown inhibited changes in Bax and mitochondrial membrane potential and events downstream of mitochondria. Activation of caspase-8 and Bid seemed to be a late event, and docetaxel was able to induce apoptosis in cells deficient in caspase-8 and Bid. p53 did not seem to be involved as a p53 null cell line was sensitive to docetaxel and an inhibitor of p53 did not inhibit apoptosis. Small interfering RNA knockdown of PUMA and Noxa also did not inhibit apoptosis. These results suggest that docetaxel induces apoptosis in melanoma cells by pathways that are dependent on activation of caspase-2, which initiates mitochondrial dependent apoptosis by direct or indirect activation of Bax.

The wide variation in sensitivity of cancer cells to TRAIL- or histone deacetylase (HDAC) inhibitor - induced apoptosis precludes successful treatment of cancer with these agents. We report here that TRAIL and SBHA synergistically induce apoptosis of melanoma cells as revealed by quantitative analysis using the normalized isobologram method. This is supported by enhanced activation of caspase-3 and cleavage of its substrates, PARP and ICAD. Co-treatment with SBHA and TRAIL did not enhance formation of the death-inducing signaling complex (DISC) and processing of caspase-8 and Bid, but potentiated activation of Bax and release of Cytochrome C and Smac/DIABLO from mitochondria into the cytosol. SBHA down-regulated Bcl-X(L), Mcl-1 and XIAP, but up-regulated Bax, Bak, and the BH3-only protein Bim(EL). Up-regulation of the latter by SBHA was attenuated by the presence of TRAIL, which was inhibitable by the pan-caspase inhibitor z-VAD-fmk. Inhibition of Bim by siRNA attenuated conformational changes of Bax, mitochondrial apoptotic events, and activation of caspase-3, leading to marked inhibition of the synergy between SBHA and TRAIL. Thus, Bim plays an essential role in synergistic induction of apoptosis by SBHA and TRAIL in melanoma.

Phenoxodiol is a chemically modified analogue of the plant hormone isoflavone with antitumour activities. In the present study, we have examined its ability to induce apoptosis in human melanoma cells and the mechanisms involved. Apoptosis was observed in Phenoxodiol-treated cells by using annexin V/propidium iodide staining and determining mitochondrial membrane potential. To determine which caspase pathways were involved in Phenoxodiol-induced apoptosis, studies were performed using specific caspase inhibitors. Western studies were performed to ascertain which proteins of the apoptosis cascade were affected to cause Phenoxodiol-induced apoptosis. We found that induction of apoptosis by Phenoxodiol was maximal at 48 h with a range of apoptosis of 12+/-4 to 48+/-5% in different melanoma lines. This apoptosis was mainly dependent on activation of caspase-3 and caspase-9. Apoptosis was associated with induction of changes in mitochondrial membrane potential and was inhibited by over-expression of Bcl-2. Variation in sensitivity to Phenoxodiol appeared related to events upstream of the mitochondria and the degree of conformational change in Bax. The p53-regulated BH3-only proteins (Bad, PUMA and Noxa) were increased in the sensitive, but not in the resistant lines, whereas Bim was increased in all the lines tested. Bim appeared, however, to be partially involved because reduction of Bim by RNA interference resulted in decreased levels of apoptosis. Together, these studies suggest that Phenoxodiol induces apoptosis of melanoma cells by induction of p53-dependent BH3 proteins (Bad, PUMA and Noxa) and the p53-independent Bim protein, resulting in activation of Bax and its downstream events.

Purpose of Review: Melanoma has proven resistant to most available chemotherapy and immunotherapy. Despite a range of different biochemical targets, most agents kill cancer cells by induction of apoptosis., Recent Findings: Investigation of this process has provided insights into the resistance mechanisms in cancer cells and to development of a range of new agents that target apoptosis pathways. These include agents which inhibit antiapoptotic B cell lymphoma-2 family proteins and inhibitor of apoptosis proteins. In addition, a range of signal pathway inhibitors have become available that are able to inhibit signal pathways known to be associated with resistance to apoptosis., Summary: Evaluation of most of these reagents are at a preclinical level but studies on some pathway inhibitors have passed from phase II into phase III studies. Similarly, evaluation of antisense reagents are at an advanced stage. These early trials show much promise and suggest this approach to development of new therapies will lead to much needed advances in treatment of this disease.

Purpose: Heterogeneous sensitivity of melanoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis may lead to outgrowth of TRAIL-resistant cells and limit successful treatment by TRAIL. The present study aims to better understand the biological characteristics of melanoma cells resistant to TRAIL-induced apoptosis., Experimental Design: We generated TRAIL-resistant melanoma cells by prolonged exposure to TRAIL and characterized the cells in terms of their sensitivity to killing induced by a panel of cytotoxic agents using biological and biochemical methods., Results: TRAIL-resistant melanoma cells are cross-resistant to apoptosis induced by another death ligand FasL, the DNA-damaging agent cisplatin, the histone deacetylase inhibitor suberic bishydroxamate, and the antimicrotubule Vinca alkaloid, vincristine. The apoptotic signaling seemed to be inhibited upstream of mitochondrial apoptotic events and was associated with decreased expression of multiple apoptotic mediators, including pro-caspase-8, Fas-associated death domain, Bid, Bim, p53, and the products of its proapoptotic target genes. Despite being resistant to apoptosis, TRAIL-resistant melanoma cells were more vulnerable to cisplatin-induced nonapoptotic cell death. This was characterized by lack of DNA fragmentation, delayed externalization of phosphatidylserine, caspase and p53 independence, and severe mitochondrial disruption, and was preceded by poly(ADP)ribose polymerase (PARP) activation and depletion of intracellular ATP, indicative of necrotic cell death. Inhibition of PARP activity partially converted the mode of cell death from necrosis to apoptosis., Conclusions: TRAIL-resistant melanoma cells are cross-resistant to apoptosis induced by various apoptotic stimuli but are more sensitive to nonapoptotic cell death induced by cisplatin. Exploration of chemotherapy-induced nonapoptotic cell death may provide an alternative strategy in overcoming resistance of melanoma cells to TRAIL-induced apoptosis.

Most anticancer agents mediate their effects through common pathways which induce apoptosis or in some cases necrosis of cancer cells. The apoptotic pathways are regulated by Bcl-2 family proteins, which include both pro- and anti-apoptotic members. Much is known about the interactions of these proteins involved in apoptosis and this information is being utilized in the development of new reagents that may be used to treat patients with cancers. The inhibitor of apoptosis family of proteins constitute a second group of proteins which inhibit the effector caspases. Reagents that inhibit their activity are also under development. Resistance of cancer cells to treatment can in many instances be attributed to activation of intracellular signal pathways involved in survival, such as the Ras-Raf-MEK-ERK1/2 or the P13K-Akt pathway. Again, much has been learned about the control of these pathways and their activation of resistance mechanisms. Inhibitors of such pathways are being evaluated in preclinical and clinical studies and are showing promise as a new class of anticancer agents. Much of the progress in future studies will likely depend on the ability to target these new treatments to particular subgroups of patients with tumor characteristics that make them responsive to the agents in question.

Protein kinase C (PKC) activation is believed to protect against apoptosis induced by death receptors. We have found however that the effect of activation of PKC on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of melanoma differs between cell lines. Pretreatment with phorbol 12-myristate 13-acetate (PMA) led to inhibition of apoptosis in the majority of the melanoma cell lines, but those with relatively low PKC epsilon expression were sensitized to TRAIL-induced apoptosis. Introduction of PKC epsilon into PKC epsilon-low cell lines reversed sensitization of the cells to TRAIL-induced apoptosis by PMA. In contrast, a dominant-negative form of PKC epsilon caused an increase in sensitivity. The changes in sensitivity to TRAIL-induced apoptosis were reflected in similar changes in conformation of Bax and its relocation from the cytosol to mitochondria. Similarly, there were concordant increases or decreases in mitochondrial release of second mitochondria-derived activator of caspase/DIABLO, activation of caspase-3, and processing of its substrates. Activation of PKC seemed to mediate its effects upstream of mitochondria but downstream of caspase-8 and Bid in that pretreatment with PMA did not cause significant changes in the expression levels of TRAIL death receptors, alterations in the levels of caspase-8 activation, or cleavage of Bid. PKC activated the anti-apoptotic extracellular signal-regulated kinase 1/2 pathway, but inhibitors of this pathway only partially reversed the protective effect of PKC against TRAIL-induced apoptosis. These results provide further insights into the variable responses of melanoma to TRAIL-induced apoptosis and may help define responsive phenotypes to treatment of melanoma with TRAIL.

Aims: Tumour necrosis factor-related apoptosis ligand (TRAIL) appears to selectively induce apoptosis in a wide range of cultured malignant cells, including melanoma. This study was designed to attempt to clarify the role of TRAIL in the biology of human melanoma., Methods: Tissue sections cut from formalin-fixed, paraffin-embedded tissue blocks of 45 primary cutaneous melanomas were tested for expression of TRAIL using immunohistochemistry. The intensity, pattern of staining and percentage of positively stained tumour cells were evaluated in each melanoma. Breslow thickness, ulcerative state, dermal mitotic rate and the presence of tumour infiltrating lymphocytes were measured/determined in each case. Median follow up for the cohort of patients was 10 months (range 1-18). Survival analysis was conducted using the Kaplan-Meier method. The level of expression of TRAIL was compared with the various histological determinants using the two-tailed Fisher's exact test and the chi2-test., Results: Overall and disease-free survival were 72 and 48%, respectively, and did not correlate with TRAIL expression. Among the pathological prognostic determinants, only mitotic rate showed a statistically significant correlation with TRAIL expression using the chi2-test (P=0.04)., Conclusion: We conclude that TRAIL expression in melanoma defines a more aggressive/proliferative phenotype, either through selection of apoptotic resistant cells or by secondary induction of other factors enhancing proliferation of more malignant cells. Analysis of a larger group of patients with longer follow-up is required to determine whether TRAIL expression correlates with survival of patients.

Ingenol 3-angelate (PEP005), one of the active ingredients in an extract from Euphorbia peplus, was shown in preclinical studies to have activity against human melanoma xenografts in nude mice. In the present study, we have tested its ability to induce the apoptosis of melanoma cells in vitro in the absence or presence of tumor necrosis factor-related apoptosis inducing ligand (TRAIL). The results showed that at relatively high concentrations (100 microg/mL), PEP005 killed melanoma cells mainly by induction of necrosis. In 20% of cell lines, evidence of apoptosis was observed. Apoptosis was caspase-dependent and associated with changes in mitochondrial membrane potential that were not inhibitable by overexpression of Bcl-2 or inhibition of caspases but were blocked by inhibition of protein kinase C (PKC). Low concentrations (1 or 10 microg/mL) of PEP005 either increased or decreased TRAIL-induced apoptosis in a cell line-dependent manner. These changes in TRAIL-induced apoptosis seemed to be due to activation of PKC and varying levels of PKC isoenzymes in different melanoma cell lines. PEP005-mediated enhancement of apoptosis seemed to be associated with low expression of the PKCepsilon isoform. These results indicate that PEP005 may enhance or inhibit sensitivity of melanoma to treatments associated with TRAIL-induced apoptosis depending on the PKC isoform content of melanoma cells.

Histone deacetylase (HDAC) inhibitors have attracted much interest because of their ability to arrest cell growth, induce cell differentiation, and in some cases, induce apoptosis of cancer cells. In the present study, we have examined a new HDAC inhibitor, suberic bishydroxamate (SBHA), for its effect on a panel of human melanoma cell lines. We report that it induces varying degrees of apoptosis in the melanoma lines but not in melanocytes and fibroblasts. Induction of apoptosis was caspase dependent and was associated with induction of changes in mitochondrial membrane permeability, which could be inhibited by overexpression of Bcl-2. The changes in mitochondria were independent of caspase activation and were associated with changes in conformation of Bax. SBHA down-regulated several key antiapoptotic proteins including X-linked inhibitor of apoptosis and the Bcl-2 family proteins, Bcl-XL and Mcl-1. In contrast, it induced up-regulation of the Bcl-2 family proapoptotic proteins, Bim, Bax, and Bak. In addition, SBHA induced relocation of the protein Bim to mitochondria and its association with Bcl-2. De novo protein synthesis was required for initiation of apoptosis in that the protein synthesis inhibitor, cycloheximide, inhibited SBHA-induced conformational changes in Bax as well as changes in mitochondrial membrane permeability and activation of caspase-3. These results suggest that SBHA induces apoptosis by changing the balance between proapoptotic and antiapoptotic proteins in melanoma cells. The protein Bim may be a key initiator of apoptosis in cells treated with SBHA.

In the present study, it was found that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-R2 protein expression did not correlate with mRNA expression in melanoma cell lines. In particular, early passage primary cultures from patients had low TRAIL-R2 protein expression compared with later passage cultures although TRAIL-R2 mRNA expression was similar in early and late passages. Similarly, cell lines made resistant to TRAIL by cultures in TRAIL had low TRAIL-R2 protein expression but normal levels of mRNA for TRAIL-R2. Expression from a luciferase reporter gene construct with the 3'-untranslated region (UTR) (but not the 5'-UTR) of TRAIL-R2 was suppressed when transfected into the TRAIL-selected (resistant) melanoma lines compared with that seen in the parental (sensitive) lines. Similar results were seen in early passage (resistant) cultures compared with late passage (sensitive) primary melanoma cultures. RNA gel shift assays demonstrated protein(s) binding to the 3'-UTR of TRAIL-R2 mRNA that were more evident in TRAIL-resistant cultures with low TRAIL-R2 protein expression. A 23-base fragment of the 3'-UTR inhibited binding of the proteins to the 3'-UTR, and a probe using this fragment bound to proteins in TRAIL-selected melanoma lines and early passage isolates of melanoma. Binding of the 3'-UTR probe to the cytosolic protein(s) was induced by exposure to TRAIL and was lost from the TRAIL selected lines 2-3 days after withdrawal of TRAIL from the cultures. These results are consistent with post-transcriptional regulation of TRAIL-R2 expression by cytosolic proteins induced by TRAIL that bind to the 3'-UTR region of TRAIL-R2 mRNA.

Staurosporine has long been used in vitro as an initiator of apoptosis in many different cell types, but the mechanism involved remains poorly understood. In the present study, we have examined the apoptosis-inducing potential of staurosporine in cultured melanoma cell lines and dissected the staurosporine-induced apoptotic signaling pathway. We report that although staurosporine activated Bax and the mitochondrial caspase-dependent apoptotic pathway, it also induced apoptosis of melanoma by caspase-independent pathways. The caspase-dependent apoptotic pathway was activated relatively soon after exposure to staurosporine and was associated with release of cytochrome c and Smac/DIABLO from mitochondria and cleavage of poly(ADP-ribose) polymerase and inhibitor of caspase-activated DNase. This pathway was inhibitable by broad caspase inhibitors. A second apoptotic pathway that appeared to be involved in late apoptotic events was caspase independent in that inhibitors of caspases did not prevent the late onset of apoptosis. Overexpression of Bcl-2 inhibited the early onset of apoptosis but not the later, caspase-independent pathway. Apoptosis-inducing factor may be responsible for the late apoptotic execution in that its translocation from mitochondria into the nucleus coincided with the late onset of apoptosis and could not be inhibited by either a pan-caspase inhibitor or overexpression of Bcl-2. Our results indicate that staurosporine is able to bypass resistance of melanoma cells to mitochondrial caspase-dependent apoptotic pathways; hence, derivatives of staurosporine may warrant further evaluation either alone or with other apoptosis-inducing agents.

TRAIL appears to be a promising anticancer agent in that it induces apoptosis in a wide range of cancer cells but not normal tissues. Sensitivity of melanoma cells to TRAIL-induced apoptosis varied considerably because of their development of various resistance mechanisms against apoptosis. We discuss in this report the potential effect of a histone deacetylase inhibitor SBHA on TRAIL-induced apoptosis. Histone deacetylase (HDAC) inhibitors regulate histone acetylation and thereby modulate the transcriptional activity of certain genes leading to cell growth arrest, cellular differentiation, and apoptosis. Suberic bishydroxamate (SBHA) is a relatively new HDAC inhibitor that induced apoptosis in the majority of melanoma cell lines through a mitochondrial and caspase-dependent pathway. This was due to its regulation of the expression of multiple proteins that are involved in either the mitochondrial apoptotic pathway (Bcl-2 family members) or the final phase of apoptosis (caspase-3 and XIAP). Co-treatment with SBHA at nontoxic doses and TRAIL resulted in a marked increase in TRAIL-induced apoptosis of melanoma, but showed no toxicity to melanocytes. SBHA appeared to sensitize melanoma to TRAIL-induced apoptosis by up-regulation of pro-apoptotic proteins in the TRAIL-induced apoptotic pathway such as caspase-8, caspase-3, Bid, Bak, and Bax, and up-regulation of the BH3 domain only protein, Bim. This, together with activated Bid, may have acted synergistically to cause changes in mitochondria. Treatment with SBHA also resulted in down-regulation of antiapoptotic members of the Bcl-2 family, Bcl-X(L) and Mcl-1, and the IAP member, XIAP. These changes would further facilitate apoptotic signaling. SBHA appeared therefore to be a potent agent in overcoming resistance of melanoma to TRAIL-induced apoptosis.

Discovery of the B cell lymphoma gene 2 (Bcl-2 gene) led to the concept that development of cancers required the simultaneous acquisition, not only of deregulated cell division, but also of resistance to programmed cell death or apoptosis. Apoptosis is arguably the common pathway to cell death resulting from a range of therapeutic initiatives, so that understanding the basis for the resistance of cancer cells to apoptosis may hold the key to development of new treatment initiatives. Much has already been learnt about the apoptotic pathways in cancer cells and proteins regulating these pathways. In most cells, apoptosis is dependent on the mitochondrial dependent pathway. This pathway is regulated by pro- and anti-apoptotic members of the Bcl-2 family, and manipulation of these proteins offers scope for a number of treatment initiatives. Effector caspases activated by the mitochondrial pathway or from death receptor signaling are under the control of the inhibitor of apoptosis protein (IAP) family. Certain proteins from mitochondrial can, however, competitively inhibit their binding to effector caspases. Information about the structure of these proteins has led to initiatives to develop therapeutic agents to block the IAP family. In addition to development of selective agents based on these two (Bcl-2 and IAP) protein families, much has been learnt about signal pathways that may regulate their activity. These in turn might provide additional approaches based on selective regulators of the signal pathways., (Copyright 2003 Wiley-Liss, Inc.)

We have previously shown that Smac/DIABLO release from mitochondria appears to be the principal pathway by which TRAIL induces apoptosis of human melanoma. We report that TRAIL-induced release of Smac/DIABLO appears to be downregulated by concomitant signaling through the MEK Erk1/2 kinase pathway and that this inhibits TRAIL-induced apoptosis. Inhibition of Erk1/2 signaling by either the MEK inhibitor U0126 or a dominant-negative mutant of MKK1 markedly sensitized melanoma cells to TRAIL-induced apoptosis. The site in the apoptotic pathway acted on by U0126 appeared to be downstream of caspase-8 and Bid but upstream of caspase-3 in that the levels of proteolytic cleavage of caspase-8 and Bid by TRAIL were similar in cells with or without exposure to U0126. Caspase-3 activation and cleavage of its substrates, PARP, ICAD and XIAP, were however increased by cotreatment with U0126. This was associated with a rapid reduction in mitochondrial transmembrane potential (MMP) and increased release of Smac/DIABLO into the cytosol. Exploration of events leading to the changes in MMP revealed an increased translocation of Bax from the cytosol to mitochondria in the presence of U0126. There was also a delayed decrease in the levels of expression of Mcl-1. Bcl-2 and Bcl-X(L). Over expression of Bcl-2 blocked TRAIL-induced apoptosis in the presence of U0126. Cytochrome c appeared not to play a major role in sensitization of melanoma to TRAIL in that caspase-9 activation was not detected in most of the cell lines. These results suggest that Erk1/2 signaling may protect melanoma cells against TRAIL-induced apoptosis by inhibiting the relocation of Bax from the cytosol to mitochondria and that this may reduce TRAIL-mediated release of Smac/DIABLO and induction of apoptosis.

At the doses used clinically, chemotherapy is believed to kill melanoma by a final common 'mitochondrial' pathway that leads to apoptosis. Similarly, several natural defence mechanisms kill melanoma by the same pathways. A corollary to the latter is that survival of melanoma in the host is due to the development of anti-apoptotic mechanisms in melanoma cells. What are these mechanisms? And how might we bypass them to improve the treatment of melanoma?

In previous studies we have shown that the sensitivity of melanoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was determined largely by the level of expression of death receptor TRAIL receptor 2 on the cells. However, approximately one-third of melanoma cell lines were resistant to TRAIL, despite expression of high levels of TRAIL receptor 2. The present studies show that these cell lines had similar levels of TRAIL-induced activated caspase-3 as the TRAIL-sensitive lines, but the activated caspase-3 did not degrade substrates downstream of caspase-3 [inhibitor of caspase-activated DNase and poly(ADP-ribose) polymerase]. This appeared to be due to inhibition of caspase-3 by X-linked inhibitor of apoptosis (XIAP) because XIAP was bound to activated caspase-3, and transfection of XIAP into TRAIL-sensitive cell lines resulted in similar inhibition of TRAIL-induced apoptosis. Conversely, reduction of XIAP levels by overexpression of Smac/DIABLO in the TRAIL-resistant melanoma cells was associated with the appearance of catalytic activity by caspase-3 and increased TRAIL-induced apoptosis. TRAIL was shown to cause release of Smac/DIABLO from mitochondria, but this release was greater in TRAIL-sensitive cell lines than in TRAIL-resistant cell lines and was associated with down-regulation of XIAP levels. Furthermore, inhibition of Smac/DIABLO release by overexpression of Bcl-2 inhibited down-regulation of XIAP levels. These results suggest that Smac/DIABLO release from mitochondria and its binding to XIAP are an alternative pathway by which TRAIL induces apoptosis of melanoma, and this pathway is dependent on the release of activated caspase-3 from inhibition by XIAP and possibly other inhibitor of apoptosis family members.

Previous studies have shown that activation of NF-kappaB can inhibit apoptosis induced by a number of stimuli. It is also known that TNF-related apoptosis-inducing ligand (TRAIL) can activate NF-kappaB through the death receptors TRAIL-R1 and TRAIL-R2, and decoy receptor TRAIL-R4. In view of these findings, we have investigated the extent to which activation of NF-kappaB may account for the variable responses of melanoma lines to apoptosis induced by TRAIL and other TNF family members. Pretreatment of the melanoma lines with the proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (LLnL), which is known to inhibit activation of NF-kappaB, was shown to markedly increase apoptosis in 10 of 12 melanoma lines with death receptors for TRAIL. The specificity of results for inhibition of NF-kappaB activation was supported by an increase of TRAIL-induced apoptosis in melanoma cells transfected with a degradation-resistant IkappaBalpha. Furthermore, studies with NF-kappaB reporter constructs revealed that the resistance of melanoma lines to TRAIL-induced apoptosis was correlated to activation of NF-kappaB in response to TRAIL. TRAIL-resistant sublines that were generated by intermittent exposure to TRAIL were shown to have high levels of activated NF-kappaB, and resistance to TRAIL could be reversed by LLnL and by the superrepressor form of IkappaBalpha. Therefore, these results suggest that activation of NF-kappaB by TRAIL plays an important role in resistance of melanoma cells to TRAIL-induced apoptosis and further suggest that inhibitors of NF-kappaB may be useful adjuncts in clinical use of TRAIL against melanoma.

In previous studies, we have shown that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) could induce varying degrees of apoptosis in approximately two-thirds of human melanoma lines. In the present study, we have examined the sensitivity of fresh isolates and early passages of melanoma cells to TRAIL-induced apoptosis from eight patients. We found that fresh isolates were relatively resistant to TRAIL-induced apoptosis and that this appeared to be associated with low TRAIL death receptor (TRAIL-R) expression. TRAIL-R expression was also undetectable in tissue sections from the same melanoma. We attempted to create a model for these findings by generation of TRAIL-resistant melanoma lines from TRAIL-sensitive lines grown for prolonged periods in TRAIL. The resulting TRAIL-resistant melanoma cell lines had low TRAIL-R expression, and sensitivity to TRAIL was increased rapidly by pretreatment with proteasome inhibitors known to inhibit activation of nuclear factor-kappaB. However, the latter treatment had no significant effect on the sensitivity of fresh isolates to TRAIL. The levels of the inhibitors of apoptosis, Flice-like inhibitory protein and Bcl-2, also did not relate to resistance to TRAIL-induced apoptosis. These results suggest that down-regulation of TRAIL-R on melanoma cells may be the primary determinant of resistance of fresh isolates to TRAIL, and the basis for this requires further investigation.

Past studies have shown that TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis in a high proportion of cultured melanoma by caspase-dependent mechanisms. In the present studies we have examined whether TRAIL-induced apoptosis of melanoma was mediated by direct activation of effector caspases or whether apoptosis was dependent on changes in mitochondrial membrane potential (MMP) and mitochondrial-dependent pathways of apoptosis. Changes in MMP were measured by fluorescent emission from rhodamine 123 in mitochondria. TRAIL, but not TNF-alpha or Fas ligand, was shown to induce marked changes in MMP in melanoma, which showed a high correlation with TRAIL-induced apoptosis. This was associated with activation of proapoptotic protein Bid and release of cytochrome c into the cytosol. Overexpression of B cell lymphoma gene 2 (Bcl-2) inhibited TRAIL-induced release of cytochrome c, changes in MMP, and apoptosis. The pan caspase inhibitor z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and the inhibitor of caspase-8 (z-Ile-Glu-Thr-Asp-fluoromethylketone; zIETD-fmk) blocked changes in MMP and apoptosis, suggesting that the changes in MMP were dependent on activation of caspase-8. Activation of caspase-9 also appeared necessary for TRAIL-induced apoptosis of melanoma. In addition, TRAIL, but not TNF-alpha or Fas ligand, was shown to induce clustering of mitochondria around the nucleus. This process was not essential for apoptosis but appeared to increase the rate of apoptosis. Taken together, these results suggest that TRAIL induces apoptosis of melanoma cells by recruitment of mitochondrial pathways to apoptosis that are dependent on activation of caspase-8. Therefore, factors that regulate the mitochondrial pathway may be important determinants of TRAIL-induced apoptosis of melanoma.

Resistance of normal cells to tumour necrosis factor related apoptosis inducing ligand (TRAIL) induced apoptosis is believed to be mediated by expression of two decoy receptors. Here we show that the expression and localisation of TRAIL receptors (TRAIL-Rs) vary between different cells and that resistance to TRAIL is mediated by different mechanisms. The decoy receptor, TRAIL-R3, appeared important in protection of endothelial cells, whereas lack of surface death receptor expression and as yet unknown intracellular inhibitor(s) of apoptosis downstream of caspase-3 may play a major role in protection of melanocytes and fibroblasts from TRAIL induced apoptosis, respectively. Differential subcellular location of decoy receptors may be an important determinant of their effectiveness in different types of normal cells.

Immune responses against human melanoma are common and are believed to influence the natural history of the disease. In particular, CD4 T cell infiltrates are associated with regression of primary melanoma and with responses to treatment with interferon-alpha2 (IFN-alpha2). Our studies have shown that CD4 T cells appear to kill melanoma by means of a member of the tumour necrosis factor (TNF) family expressed on their surface and called TNF related apoptosis inducing ligand (TRAIL). Moreover, sensitivity to TRAIL also predicts responsiveness of melanoma to CD4 T cells. TRAIL is not expressed on resting lymphocytes but is expressed at high levels after exposure to IFN-alpha2 and on activated T cells. Lymphocytes from melanoma patients in early stages of the disease show high levels of expression after exposure to IFN-alpha2 and IFN-gamma but expression was less on lymphocytes from stage IV patients. This may be due to factors from melanoma cells in that supernatants from some melanoma cultures suppressed IFN-alpha2 upregulation of TRAIL. Sensitivity of melanoma cells to TRAIL can be increased by inhibition of the activation of NF-kappaB and anti-apoptotic events downstream of NF-kappaB. These results suggest that TRAIL may be an important mediator of responses against melanoma induced by immunotherapy or by treatment with IFN-alpha2 and interleukin-2. Studies on surgical biopsies of melanoma however show that fresh isolates appear less sensitive to TRAIL-induced apoptosis and effective therapy may involve combinations with other agents.