Lothar F. Fecker

Resistance of melanoma cells to TRAIL does not result from upregulation of antiapoptotic proteins by NF-kappaB but is related to downregulation of initiator caspases and DR4.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention as a novel anticancer agent. However, its efficiency may be diminished by occurring resistance in cancer cells. The mechanisms of TRAIL resistance in melanoma are still unsolved. Here we show for the first time that TRAIL-induced activation of NF-κB occurs in apoptosis-sensitive melanoma cell lines through TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4), whereas TRAIL failed to activate nuclear factor kappa B (NF-κB) in melanoma cells positive only for TRAIL receptor 2/death receptor 5 (TRAIL-R2/DR5). However, activation of NF-κB by TRAIL was not associated with enhanced expression of antiapoptotic factors: cellular FLICE-inhibitory protein (c-FLIP), Bcl-xL, X-linked inhibitor of apoptosis protein (XIAP), Survivin, Livin. Rather in one of the cell lines, TRAIL induced the downregulation of DR4. In an established cell culture model for TRAIL resistance and regained TRAIL sensitivity, resistance was neither associated with increased NF-κB activity by TRAIL nor by an increased expression of antiapoptotic proteins. However, significant downregulation of caspase-8, caspase-10 and of DR4 was characteristic for TRAIL-resistant, DR4-positive melanoma cells, and regained TRAIL sensitivity coincided with re-expression of these factors. Sensitivity was also largely retained after their exogenous overexpression. Thus, initiator caspases and DR4 rather than NF-κB may control melanoma cell sensitivity to TRAIL, and strategies, which result in their upregulation, may be useful for enhancement of TRAIL sensitivity.

CD95/Fas signaling in human melanoma cells: conditional expression of CD95L/FasL overcomes the intrinsic apoptosis resistance of malignant melanoma and inhibits growth and progression of human melanoma xenotransplants

The significance of CD95/Fas ligand expression by melanoma cells has remained a controversial matter in recent years. On the other hand, CD95 activation may represent a powerful tool for eliminating tumor cells. Here, we demonstrate expression of CD95 in 15/17 human melanoma cell lines analysed, but complete lack of CD95 ligand (CD95L). Overexpression of CD95 in a tetracycline-inducible expression system enhanced melanoma cell sensitivity to CD95 ligation but was unable to trigger apoptosis by itself. In clear contrast, all melanoma cells tested responded with increased apoptosis to conditional expression of CD95L (2–10-fold), both after transient and after stable transfection. Activation of caspase-8, Bid cleavage, cytochrome c release and caspase-3 activation followed after CD95L induction indicating a functional CD95-signaling cascade. CD95L was also able to enhance the proapoptotic effect of chemotherapeutics applied in parallel. Nude mouse experiments revealed that tumorigenicity was lost when melanoma xenografts were triggered to express CD95L. In addition, further progression of pre-existing melanomas was inhibited and even regression was seen after induction of CD95L expression. Due to these data, transfection of CD95L proofs as a highly efficient tool against melanoma cells in vitro and in vivo, and targeted expression of CD95L may thus represent a suitable strategy for melanoma therapy.

Caspase-independent induction of apoptosis in human melanoma cells by the proapoptotic Bcl-2-related protein Nbk/Bik

The proapoptotic BH3-only protein natural born killer / Bcl-2 interacting killer (Nbk / Bik) has been described to inhibit Bcl-2 and Bcl-xL, thereby supporting the death promoting ability of Bax. In order to evaluate its function in melanoma, we investigated the response after Nbk / Bik overexpression in cultured human melanoma cells and in a melanoma mouse model. Untransfected melanoma cell lines expressed Nbk / Bik only weakly at the mRNA and protein level. Conditional expression of Nbk / Bik by applying the inducible tetracycline-responsive expression system triggered apoptosis and enhanced sensitivity to proapoptotic stimuli as to agonistic CD95 activation and to chemotherapeutics etoposide, doxorubicin and pamidronate. For investigating the effects of Nbk / Bik in vivo, stably transfected melanoma cells were subcutaneously injected into nude mice. Significantly delayed tumor growth was the result when mice received doxycycline for induction of Nbk / Bik expression. By investigating the mechanism of Nbk / Bik-induced cell death, typical hallmarks of apoptosis such as DNA fragmentation and chromatin condensation were seen after induction. Interestingly, no indications for cytochrome c release and caspase processing were found, and selective caspase inhibition remained without effect. These data indicate the high potential of Nbk / Bik in regulating apoptosis in melanoma by a caspase-independent pathway and may corroborate the potency of novel antimelanoma strategies based on activation of BH3-only proteins such as Nbk / Bik.

Apoptosis pathways and oncolytic adenoviral vectors: promising targets and tools to overcome therapy resistance of malignant melanoma

Abstract: In the last decades melanoma incidence has been increasing worldwide, while mortality remained on a high level. Until now, there is no suitable therapy for metastasized melanoma, which could lead to a significant increase in overall survival. Apoptosis deficiency is supposed to be a critical factor for therapy resistance, and previous work has characterized the basic mechanisms of apoptosis regulation in melanoma. Genes and strategies suitable for efficient induction of apoptosis in melanoma cells were identified, which are based on proapoptotic Bcl‐2 proteins (Bcl‐xS, Bcl‐xAK, Bik/Nbk and Bax) as well as on tumor necrosis factor (TNF)‐related death ligands (CD95L/Fas ligand and TNF‐related apoptosis‐inducing ligand, TRAIL). Proapoptotic genes may be employed in improved gene therapeutic strategies, based on conditional oncolytic adenoviral vectors.

Enhanced Death Ligand-Induced Apoptosis in Cutaneous SCC Cells by Treatment with Diclofenac/Hyaluronic Acid Correlates with Downregulation of c-FLIP

Actinic keratosis (AK) occurs on sun-exposed skin and may progress to invasive squamous cell carcinoma (SCC). As for its topical treatment, diclofenac/hyaluronic acid (HA) has been recently approved. The NSAID diclofenac is an inhibitor of COX-2; however, its mode of action in cutaneous epithelial cancer cells is largely unknown. Here, the effects of diclofenac/HA were investigated in relation to death ligand-mediated apoptosis (TNF-alpha, TRAIL, and CD95 activation). Whereas diclofenac/HA only moderately induced apoptosis by itself, it resulted in pronounced enhancement of death ligand-mediated apoptosis in sensitive SCC cell lines (3/4). Apoptosis was associated with activation of initiator caspases of the extrinsic pathway (caspase-8/caspase-10). Furthermore, death ligand and diclofenac/HA-mediated apoptosis were blocked by the same caspase inhibitors, indicating related pathways. The proapoptotic effects of diclofenac/HA appeared independent of the p53 pathway. Also, upregulation of death receptors appeared less important; however, strong downregulation of c-FLIP isoforms was seen after diclofenac/HA treatment. The crucial role of c-FLIP was proven through overexpression and knockdown experiments. Thus, induction of apoptosis appears to be highly characteristic of the mode of action of diclofenac/HA, and the therapeutic effect may be related to sensitization of neoplastic keratinocytes for death ligand-induced apoptosis.

Conditional expression of exogenous Bcl-XS triggers apoptosis in human melanoma cells in vitro and delays growth of melanoma xenografts

The Bcl‐2‐related proteins Bcl‐XL and Bcl‐XS represent alternative splice products and exert opposite activities in the control of apoptosis, but their significance for melanoma is not yet clear. Applying the tetracycline‐inducible expression system Tet‐On, we found overexpression of Bcl‐XS by itself sufficient to induce apoptosis in vitro in stably transfected human melanoma cell lines. Combination with proapoptotic agents such as etoposide, pamidronate, and ceramide resulted in additive proapoptotic effects, whereas Bcl‐XL protected from apoptosis caused via CD95/Fas stimulation. In nude mice growth of melanoma xenotransplants derived from stably transfected cells was significantly reduced after induction of Bcl‐XS by doxycycline. Our results indicate that Bcl‐X proteins are of major importance for control of apoptosis in malignant melanoma.

Apoptosis Induction by SAHA in Cutaneous T-Cell Lymphoma Cells Is Related to Downregulation of c-FLIP and Enhanced TRAIL Signaling

Suberoylanilide hydroxamic acid (SAHA) has been approved for the treatment of cutaneous T-cell lymphoma (CTCL), but its mode of action remained largely elusive. As shown here in four CTCL cell lines, loss of cell viability correlated with significant time- and dose-dependent induction of apoptosis, whereas cytotoxicity was less pronounced. Both extrinsic and intrinsic apoptosis pathways were activated, as seen by processing of initiator caspases 8 and 9, loss of mitochondrial membrane potential, and cytochrome c release. Characteristically, antiapoptotic mediators such as Mcl-1, XIAP, survivin, and c-FLIP were downregulated. Consistent with its critical function, c-FLIP overexpression resulted in a significant decrease of SAHA-mediated apoptosis. Enhanced sensitivity to TRAIL (TNF-related apoptosis-inducing ligand) and enhanced TRAIL signaling was seen in CTCL cell lines with high sensitivity, whereas cell lines with moderate response were characterized by downregulation of TRAIL-R2 and weaker TRAIL expression. Comparable proapoptotic responses to SAHA and to the combination with TRAIL were seen in ex vivo tumor T cells of CTCL patients. Thus, activation of extrinsic apoptosis pathways, related to c-FLIP downregulation and enhanced TRAIL signaling, appeared as characteristic for CTCL cell responsiveness to SAHA. An improved understanding of the pathways may facilitate its targeted use and the selection of suitable combinations.

A novel Bcl-x splice product, Bcl-xAK, triggers apoptosis in human melanoma cells without BH3 domain

Pro- and antiapoptotic proteins of the large Bcl-2 family are critical regulators of apoptosis via the mitochondrial pathway. Whereas antiapoptotic proteins of the family share all four Bcl-2 homology domains (BH1–BH4), proapoptotic members may lack some of these domains, but all so far described proapoptotic Bcl-2 proteins enclose BH3. The bcl-x gene gives rise to several alternative splice products resulting in proteins with distinct functions as the antiapoptotic Bcl-xL and proapoptotic Bcl-xS. Here, we describe a novel Bcl-x splice product of 138 amino acids termed Bcl-xAK (Atypical Killer), which encloses the Bcl-2 homology domains BH2 and BH4 as well as the transmembrane domain, but lacks BH1 and BH3. Weak endogenous expression of Bcl-xAK was seen in melanoma and other tumor cells. Interestingly, its overexpression by applying a tetracycline-inducible expression system resulted in significant induction of apoptosis in melanoma cells, which occurred in synergism with drug-induced apoptosis. After exogenous overexpression, Bcl-xAK was localized both in mitochondrial and in cytosolic cell fractions. By these findings, a completely new class of Bcl-2-related proteins is introduced, which promotes apoptosis independently from the BH3 domain and implies additional, new mechanisms for apoptosis regulation in melanoma cells.

Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication‐competent CD95L adenovirus

Please cite this paper as: Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication‐competent CD95L adenovirus. Experimental Dermatology 2010; 19: e56–e66.

Activation of mitochondrial apoptosis pathways in cutaneous squamous cell carcinoma cells by diclofenac/hyaluronic acid is related to upregulation of Bad as well as downregulation of Mcl-1 and Bcl-w.

Actinic keratosis (AK) is characterized by high prevalence and the risk to proceed to squamous cell carcinoma (SCC). Cyclooxygenase‐2 (COX‐2)‐mediated prostaglandin E2 (PGE 2) synthesis has been reported in AK and SCC, and the COX inhibitor diclofenac in hyaluronic acid (diclofenac/HA) was approved for AK therapy. Its mode of action, however, remained to be unravelled. In the present study, diclofenac resulted in reduced PGE 2 levels in apoptosis‐sensitive cutaneous SCC cell lines (SCL‐II, SCC‐12, SCC‐13) whereas no PGE 2 and no COX‐2 expression was detectable in a SCC cell line resistant to apoptosis induction (SCL‐I). Activation of mitochondrial apoptosis pathways was evident in SCC cells owing to loss of the mitochondrial membrane potential and release of the mitochondrial factors cytochrome c and apoptosis‐inducing factor. Characteristic proapoptotic changes at the level of Bcl‐2 proteins occurred in sensitive cells, as upregulation of Bad and downregulation of Mcl‐1 and Bcl‐w. In contrast, Bad was already high, and Mcl‐1 and Bcl‐w were already low in resistant SCL‐I, even without treatment, which may be explained by the lack of PGE 2. An antiapoptotic downregulation of proapoptotic Bcl‐2 proteins Noxa and Puma was, however, also seen in SCL‐I, suggesting here pathways independent of COX‐2. The regulations of Mcl‐1 and Bad were also reproduced in SCC cells by the more selective COX‐2 inhibitor celecoxib, thus further underlining the specific role of COX‐2. The findings illuminate the mode of action of diclofenac/HA in SCC cells as well as principles of their resistance, which may allow further adaptation and improvement of the new therapy.