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Melanoma is a highly aggressive malignant tumor with an exceptional ability to develop resistance and no curative therapy is available for patients with distant metastatic disease. The inhibitor of apoptosis protein (IAP) family has been related to therapy resistance in cancer. We examined the importance of the IAPs in the resistance to the commonly used chemotherapeutic agent dacarbazine (DTIC) and the apoptosis inducer TRAIL (TNF-related apoptosis inducing ligand) in malignant melanoma. The data presented show that the expression of IAPs is universal, concomitant and generally high in melanoma cell lines and in patient samples. Depleting IAP expression by siRNA tended to reduce cell viability, with XIAP reduction being the most efficient in all four cell lines examined (FEMX-1, LOX, SKMEL-28 and WM115). The combined treatment of XIAP siRNA and DTIC showed a weak improvement in two of four cell lines, while all four cell lines showed enhanced sensitivity towards TRAIL (AdhCMV-TRAIL) after XIAP depletion. In addition, cIAP-1, cIAP-2 and survivin down-regulation sensitized to TRAIL treatment in several of the cell lines. Cells exposed to TRAIL and XIAP siRNA showed increased DNA-fragmentation and cleavage of Bid, procaspase-8, -9, -7 and -3 and PARP, and change in the balance between pro- and anti-apoptotic proteins, indicating an enhanced level of apoptosis. Furthermore, the combined treatment reduced the ability of melanoma cells to engraft and form tumors in mice, actualizing the combination for future therapy of malignant melanoma.

Background: Currently, dacarbazine (DTIC) is the only approved systemic treatment for metastatic malignant melanoma. However, the modest treatment effect encourages studies on novel therapeutic molecules, delivery systems and combination therapies. Full-length TRAIL, delivered from an adenoviral vector (Ad-hTRAIL), was studied in combination with DTIC or the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) in human melanoma cell lines., Methods: The cytotoxic potential of the combination treatments was assessed by cell viability measurements and CalcuSyn analysis. Involvement of apoptosis was analyzed by TUNEL staining, mitochondrial membrane potential measurements, and activation and expression levels of caspases and other mediators of apoptosis., Results: Ad-hTRAIL in combination with DTIC or SAHA resulted in additive or synergistic growth inhibition compared to each treatment used as single agent. Both combinations augmented apoptosis, which was mediated through the death receptor (DR) pathway by enhanced activation of caspase-8, and through increased loss of mitochondrial integrity. Provoked cleavage of Bid, which bridges the extrinsic and intrinsic apoptosis pathways, and downregulation of the anti-apoptotic mediators Bcl-X(L), Mcl-1 and XIAP (but not Bcl-2) were critical contributing factors. Increased levels of DR4 and DR5 were not a common underlying mechanism as DTIC did not affect the levels of either of the receptors. However, SAHA-induced expression of DR4 may have reduced the TRAIL resistance in the SKMEL-28 cell line., Conclusion: Administration of Ad-hTRAIL in combination with DTIC or SAHA enhances apoptosis in human melanoma cell lines, and suggests that the therapeutic potential of such treatment strategies should be further evaluated for possible clinical use.

Current treatment regimens for patients with metastatic melanoma are not curative, and new treatment strategies are needed. One possible approach is targeted treatment using the tyrosinase promoter for melanoma-specific expression of genes delivered by adenoviral (Ad) vectors. In this study, a vector with the human minimal tyrosinase promoter and two human enhancer elements (2hE-hTyrP) was compared with different hybrid promoter constructs, containing tyrosinase regulatory sequences and the viral simian virus 40 (SV40) promoter. The tissue specificity of the first-generation vectors was measured by enhanced green fluorescence protein (EGFP) reporter flow cytometry in 12 human melanoma and nonmelanoma cell lines. In the melanotic melanoma cells, the activity of the 2hE-hTyrP promoter was comparable with the activity of the cytomegalovirus promoter, and the background expression levels obtained in the nonmelanoma cell lines confirmed the strict tissue-specific property of this promoter. The hybrid SV40-based promoters were effective, but no tissue specificity was observed even after the inclusion of tyrosinase enhancer elements identical to the elements used in the 2hE-hTyrP promoter. The in vivo tissue specificity of the 2hE-hTyrP vector was demonstrated in subcutaneous xenografted tumors by ex vivo detection of EGFP fluorescence with the IVIS Imaging equipment and fluorescence microscopy visualizing the in situ EGFP expression in tumor sections. The tyrosinase mRNA level in the 12 cell lines was measured by quantitative real-time RT-PCR, and the expression levels reliably reflected to what extent the 2hE-hTyrP promoter could drive the gene expression in the individual cell lines. In conclusion, the human tyrosinase promoter fused to two human tyrosinase enhancers (2hE-hTyrP) can be used for efficient tissue-specific expression from first-generation Ad vectors in melanoma cell lines both in vitro and in vivo, as predicted by the quantitative tyrosinase mRNA levels in the melanoma and nonmelanoma cell lines tested.

Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.

Entrapment and degradation of transfecting DNA in endocytic vesicles often hampers the use of lipidic vectors for gene delivery purposes. Photochemical internalisation (PCI) is a technology for achieving light-induced release of DNA trapped inside these vesicles, and therefore represents a way of overcoming the endocytic membrane barrier and improving gene transfer. The technology is based on utilising photosensitizers which localise in the membranes of endocytic vesicles, causing photochemical damages that rupture the vesicles upon illumination. The purpose of this work was to study the effect of PCI on transfection mediated by the cationic lipid N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (betaAE-DMRIE), with or without the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). It was shown that PCI has no effect on betaAE-DMRIE mediated transfection, whereas it significantly enhances transfection mediated by the combination of betaAE-DMRIE and DOPE. The effect of PCI was highly dependent on the timing of illumination relative to the time of DNA delivery, both regarding the sequence of, and the time between, these two treatments.

Most synthetic gene delivery vectors are taken up in the cell by endocytosis, and inefficient escape of the transgene from endocytic vesicles often is a major barrier for gene transfer by such vectors. To improve endosomal release we have developed a new technology, named photochemical internalization (PCI). PCI is based on photochemical reactions initiated by photosensitizing compounds localized in endocytic vesicles, inducing rupture of these vesicles upon light exposure. PCI constitutes an efficient light-inducible gene transfer method in vitro, which potentially can be developed into a site-specific method for gene delivery in in vivo gene therapy. In this paper the principle behind the PCI technology and the effect of PCI on transfection with different synthetic gene delivery vectors are reviewed. PCI treatment by the photosensitizer aluminum phthalocyanine (AlPcS2a) strongly improves transfection mediated by cationic polymers (e.g., poly-L-lysine and polyethylenimine), while the effect on transfection with cationic lipids is more variable. The timing of the light treatment relative to the transfection period was also important, indicating that release of the DNA from early endosomes is important for the outcome of PCI-induced transfection. The possibilities of using PCI as a technology for efficient, site-specific gene delivery in in vivo gene therapy is discussed.

A main issue for further clinical progress of cancer gene therapy is to develop technologies for efficient and specific delivery of therapeutic genes to tumor cells. In this work, we describe a photochemical treatment that substantially improves gene delivery by adenovirus, one of the most efficient gene delivery vectors known. Transduction of two different cell lines was studied by microscopy, flow cytometry, and an enzymatic assay, employing a beta-galactosidase-encoding adenovirus. The photochemical treatment induced a >20-fold increase in gene transduction, compared with conventional adenovirus infection, both when measured as the percentage of cells transduced, and when measured as the total beta-galactosidase activity in the cell population. The effect was most pronounced at lower virus doses, where in some cases the same transduction efficiency could be achieved with a 20 times lower virus dose than with conventional infection. Photochemical treatments are already in clinical use for cancer therapy, and generally are very specific and have few side effects. The photochemical internalization technology described thus has a clear potential for improving both the efficiency and the specificity of gene delivery in cancer gene therapy, making it possible to achieve efficient site-specific in vivo gene delivery by adenoviral vectors.