Abstract
Resistance of tumor cells to cisplatin is a common feature frequently encountered during chemotherapy against melanoma caused by various known and unknown mechanisms. To overcome drug resistance toward cisplatin, a targeted treatment using alternative agents, such as proteasome inhibitors, has been investigated. This combination could offer a new therapeutic approach. Here, we report the biological effects of proteasome inhibitors on the parental cisplatin-sensitive MeWo human melanoma cell line and its cisplatin-resistant MeWo(cis1) variant. Our experiments show that proteasome inhibitor treatment of both cell lines impairs cell viability at concentrations that are not toxic to primary human fibroblasts in vitro. However, compared with the parental MeWo cell line, significantly higher concentrations of proteasome inhibitor are required to reduce cell viability of MeWo(cis1) cells. Moreover, whereas proteasome activity was inhibited to the same extent in both cell lines, IkappaBalpha degradation and nuclear factor-kappaB (NF-kappaB) activation in MeWo(cis1) cells was proteasome inhibitor independent but essentially calpain inhibitor sensitive. In support, a calpain-specific inhibitor impaired NF-kappaB activation in MeWo(cis1) cells. Here, we show that cisplatin resistance in MeWo(cis1) is accompanied by a change in the NF-kappaB activation pathway in favor of calpain-mediated IkappaBalpha degradation. Furthermore, combined exposure to proteasome and calpain inhibitor resulted in additive effects and a strongly reduced cell viability of MeWo(cis1) cells. Thus, combined strategies targeting distinct proteolytic pathways may help to overcome mechanisms of drug resistance in tumor cells.
Highlights
Melanoma is a malignant neoplasia of melanoblastic origin that is the leading cause of death from cutaneous malignant disease [1]
To determine the cytotoxic and cytostatic effects of proteasome inhibition on human melanoma cells, MeWo or MeWocis1 cells were exposed to different concentrations of proteasome inhibitor BSc2118 for 48 hours (Fig. 1A) and the viability of the cells was determined by crystal violet assay
The IC50 for BSc2118 determined was 30 nmol/L for MeWo cells and 70 nmol/L for MeWocis1 cells (Fig. 1A). Both melanoma cell lines are sensitive to BSc2118, considerably larger amounts of the inhibitor are required to induce a reduction of cell viability in MeWocis1 cells
Summary
Melanoma is a malignant neoplasia of melanoblastic origin that is the leading cause of death from cutaneous malignant disease [1]. Combination of conventional chemotherapeutics with novel biological agents, such as proteasome inhibitors, need to be studied carefully. In this context, proteasome inhibitors have been described that induce cell cycle arrest and apoptosis, and that could sensitize malignant cells to proapoptotic effects of conventional chemotherapeutics [6, 7]. The proteasome system is involved in various pathways, such as regulation of transcription, apoptosis, and cell cycle. The proteasome activates nuclear factor-nB (NF-nB) by degrading its inhibitory proteins (InB). Previous studies indicate that in this context, NF-nB could be activated in a proteasome-ubiquitin-independent pathway due to calpain-dependent InBa degradation [8]. Calcium-dependent proteases that cleave a specific subset of cellular proteins, including cytoskeletal proteins, membrane receptors, and many transcription factors [9]
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