These results show that fenretinide and velcade induce ER stress in melanoma cells, that the antibiotic PDI inhibitor bacitracin increases cell death as a consequence of ER stress and that these effects of bacitracin result from its ability to inhibit PDI activity. Although both fenretinide and velcade induced similar ER stress responses, the mechanism of action of these drugs is very different. Fenretinide induces stress via an oxidative stress mechanism (3
) which, so far, has not been characterised. Conversely, velcade is a specific inhibitor of the 26S-proteasome; however, the central role of the proteasome in most cellular functions gives velcade the ability to inhibit cellular function on a broad scale resulting in ER stress, but with the time-course and end result, in terms of cell arrest or apoptosis, varying depending on cell type (32
). Cancer cells, particularly melanoma and leukaemia, are more sensitive to velcade than normal cells (16
). Although leukaemia cells are also more sensitive than normal cells to fenretinide (34
), this is not true with respect to ovarian cancer/ normal ovarian epithelial cells (35
) and the normal melanocytes used in this study were relatively sensitive to fenretinide; preliminary data for the normal melanocytes suggested that these cells accumulated fenretinide more readily than melanoma cells (Armstrong, unpublished data) and such differences in uptake is one mechanism that could explain cell-type-specific variability in fenretinide sensitivity.
Despite the sensitivity of normal melanocytes to fenretinide, bacitracin treatment did not increase the response to fenretinide or to velcade. This is in clear contrast to the melanoma cells in which bacitracin produced a dose-dependent increase in cell death in response to fenretinide or velcade which was synergistic with fenretinide and additive with velcade across a broad dose-range. Therefore, these results suggest that inhibiting PDI activity may offer a novel mechanism to increase the therapeutic benefit of chemotherapeutic drugs for treating metastatic melanoma, particularly in the context of ER stress-inducing agents. The potential for enhancing the activity of velcade is particularly significant as this drug has good clinical activity in myeloma and is being investigated for use in a range of other cancers (36
). Furthermore, velcade has shown promise in combination with existing chemotherapeutic drugs (32
) and enhances responses of melanoma cells to temozolomide in vitro
). The ability to increase cancer-cell-specific effects of velcade with PDI inhibitors would be a powerful addition to the chemotherapeutic arsenal. However, since the clinical use of bacitracin is limited by its nephrotoxicity (38
) and low membrane permeability (31
), it is important to develop safer and more-effective alternatives. This can only be done with adequate knowledge of how bacitracin achieves its effects. Bacitracin did indeed reduce PDI activity in A375 cells; however, bacitracin can have PDI-independent effects (40
), has metal-binding activity and the commercial product can comprise a mixture of bacitracins differing in amino-acid sequence and N-terminal structure (27
In the absence of purified bacitracins and data to link PDI inhibition to specific bacitracins, it is critical to verify that the ability of ‘bacitracin’ to increase velcade or fenretinide-induced cell death is a result of PDI inhibition. As predicted, over-expression of P4HB in melanoma cells increased PDI activity and substantially reduced the ability of bacitracin to increase apoptosis in combination with either fenretinide or velcade, presumably by competing with endogenous PDIs for binding to the PDI-inhibition function of bacitracin. The mutant P4HB used as a negative control for this experiment lacked PDI activity as a result of cysteine to serine mutations in both active PDI sites (11
); the fact that this mutant P4HB construct, despite being over-expressed to similar levels as wild-type P4HB, did not abrogate the potentiation of fenretinide- or velcade-induced apoptosis by bacitracin is strong evidence that inhibiting PDI activity reduces the ability of cells to survive ER stress. These results also show the importance of PDI activity in facilitating the survival of cells exposed to ER stress. In melanoma cells, PDIs have been reported to be expressed at a higher level than normal melanocytes (41
) and the expression of PDI family proteins correlates with cancer invasion, metastasis and drug resistance in other tumor types (43
). Although recent studies indicate that the sensitivity of melanoma cells to TRAIL-induced apoptosis may be increased by modulation of the UPR (46
), the present results suggest that PDI inhibition can be used as a more-general mechanism to down-regulate homeostatic stress responses and drive apoptosis.
The results of this study provide proof-of-principle for the concept that that ER-stress-induced apoptosis of melanoma cells can be enhanced using PDI inhibitors. Furthermore, the data imply that potent, small-molecule PDI inhibitors designed to bind to the CXXC motif of the PDI active site may have significant potential as powerful tools for enhancing the efficacy of chemotherapy in a wide range of cancers.